Modified drugs for use in liposomal nanoparticles

ABSTRACT

Drug derivatives are provided herein which are suitable for loading into liposomal nanoparticle carriers. In some preferred aspects, the derivatives comprise a poorly water-soluble drug derivatized with a weak-base moiety that facilitates active loading of the drug through a LN transmembrane pH or ion gradient into the aqueous interior of the LN. The weak-base moiety can optionally comprise a lipophilic domain that facilitates active loading of the drug to the inner monolayer of the liposomal membrane. Advantageously, LN formulations of the drug derivatives exhibit improved solubility, reduced toxicity, enhanced efficacy, and/or other benefits relative to the corresponding free drugs.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.12/993,482 filed on Feb. 14, 2011, which is a 35 U.S.C. 371 nationalstage application of PCT/IB2009/006532 filed on May 26, 2009, whichclaims the benefit of U.S. provisional patent application No. 61/055,929filed on May 23, 2008, the contents of each are herein incorporated byreference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to methods of chemicallymodifying drugs that are resistant or incapable of being encapsulated inliposomes to form derivatives that can be efficiently loaded intoliposomal nanoparticles (LN) exhibiting a transmembrane pH or iongradient. In some preferred aspects, the derivatives are pro-drugs thatare readily converted to the free drug upon release from the LN. Theinvention also relates to drug derivatives made according to methods ofthe invention, LN formulations and pharmaceutical compositionscomprising such derivatives, and methods of making and using the same.

BACKGROUND

Many existing drug discovery strategies are predicated on finding‘druggable’ compounds that are water-soluble and bioavailable. As aresult, newly discovered compounds with poor solubility and limitedbioavailability rarely advance to lead status, often despite havingpromising therapeutic properties.

A variety of drug formulations and delivery methods have been developedin an effort to overcome the limitations of non-druggable compounds.Liposomal nanoparticles (LN) are a leading drug delivery system for thesystemic (intravenous) administration of drugs, and there are a numberof liposomal drugs currently on the market and in clinical trials. LNgenerally have low toxicity and can be designed to provide a wide rangeof beneficial pharmaceutical properties, such as improved serumhalf-life, bioavailability, permeability, and the like. LN formulationshave been particularly successful in connection with chemotherapeuticagents, which have limited efficacy when administered in theirconventional (free) form due to their low aqueous solubility, shortserum half-life, and indiscriminate accumulation in normal and diseasetissues alike.

Long-circulating LN typically have diameters of about 100 nm or less,and remain in the blood circulation for extended periods of time. Theextended lifespan of long-circulating LN allows them to accumulate at ornear sites of infection, inflammation, tumor growth, and otherdisease-associated drug targets. This accumulation is facilitated by thelocal structure of the vasculature in these regions (referred to as“leaky” vasculature), characterized by large pores through whichliposomes can reach therapeutic targets (Jain, Microcirculation, 4: 1-23(1997), Hobbs et al., Proc. Natl. Acad. Sci. USA, 95: 4607-4612 (1998)).Stable association of a chemotherapeutic agent or other drug withlong-circulating LN can therefore increase the amount of the drug thatreaches therapeutic targets, prolong the exposure of the targets totherapeutic levels of the drug through controlled (sustained) releasefrom the LN, and reduce accumulation in healthy, non-targeted tissues,thereby increasing effectiveness and reducing toxicity. In the case ofsolid tumors, LN formulations of chemotherapeutic agents have yieldeddramatic improvements in therapeutic index, tolerability, efficacy, andother properties, in both animal models and clinical studies.

The application of LN technology to a drug of interest requires the drugto be amenable to being loaded in a liposomal carrier and released at anappropriate rate at or near therapeutic targets. The ability to load adrug into liposomes depends on the chemical properties of the drug, theliposomal membrane, and the interior environment of the liposome. Ingeneral, both water soluble and lipid soluble drugs can be loaded intoliposomes using passive loading techniques that rely on the associationof water soluble drugs with the polar phospholipids lining the innerliposomal membrane and/or the aqueous liposomal interior, and theassociation of lipid soluble drugs with the lipid bilayer. However, manyuseful drugs have more complex solubility profiles that are lessamenable to passive loading methods.

One approach for loading poorly soluble drugs into liposomes is tomodify the drug to facilitate passive loading. For example, liposomalformulations have been developed in which taxanes are modified by theaddition of a hydrocarbon chain containing an electronegative“hydrolysis-promoting group” (HPG) to form fatty acid derivatives withenhanced solubility in the lipid bilayer, as described in U.S. Pat. No.6,482,850 and related applications. However, passive loading methodsgenerally have poor loading efficiencies and produce liposomes with poordrug retention and release, limiting the utility of the resultingformulations.

To overcome limitations related to passive loading, several activeloading techniques have been developed that allow drugs to be loadedwith high efficiency and retention. A particularly effective approachinvolves loading of drugs that are weak bases by forming a pH gradientacross the liposomal membrane to produce liposomes with an acidicliposomal interior and an exterior environment with higher pH than theliposome interior (e.g. neutral pH) (e.g., Maurer, N., Fenske, D., andCullis, P. R. (2001) Developments in liposomal drug delivery systems.Expert Opinion in Biological Therapy 1, 923-47; Cullis et al., BiochimBiophys Acta., 1331: 187-211 (1997); Fenske et al., Liposomes: Apractical approach. Second Edition. V. Torchilin and V. Weissig, eds.,Oxford University Press, p. 167-191 (2001)). Weakly basic drugs canexist in two co-existing (equilibrium) forms; a charge-neutral(membrane-permeable) form and a charged/protonated (membraneimpermeable) form. The neutral form of the drug will tend to diffuseacross the liposome membrane until the interior and exteriorconcentrations are equal. However, an acidic interior environmentresults in protonation of the neutral form, thereby driving continueduptake of the compound trapping it in the liposome interior. Anotherapproach involves the use of metal ion gradients (e.g. Cheung B C, Sun TH, Leenhouts J M, Cullis P R: Loading of doxorubicin into liposomes byforming Mn²⁺-drug complexes. Biochim Biophys Acta (1998) 1414:205-216).The metal ion concentration is high in the liposome interior; theexterior environment is metal ion free. This loading method relies thesame basic principles as the pH gradient technique. The neutral form ofthe weak base drug can permeate across the membrane and is retained inthe aqueous interior of the liposomes through formation of a drug-metalion complex. In this case drug-metal ion complex formation drives thecontinued uptake of the drug.

Some anticancer and antimicrobial drugs, such as vincristine,vinorelbine, doxorubicin, ciprofloxacin and norfloxacin, can be readilyloaded and stably retained in LN using pH gradient active loadingtechniques (e.g., Drummond et al., Pharmacol. Rev., 51: 691-743 (1999),Cullis et al., Biochim Biophys Acta., 1331: 187-211 (1997); Semple etal., J. Pharm. Sci., 94(5): 1024-38 (2005)). However, a number ofclinically important drugs are not weak bases and are thus not amenableto such active loading techniques (e.g., Soepenberg et al., European J.Cancer, 40: 681-688 (2004)). For example, many anticancer drugs,including certain taxane-based drugs (e.g., paclitaxel and docetaxel),and podophyllotoxin derivatives (e.g., etoposide) cannot readily beformulated as LN using standard methods.

Taxotere® (docetaxel) and Taxol® (paclitaxel) are the most widelyprescribed anticancer drugs on the market, and are associated with anumber of pharmacological and toxicological concerns, including highlyvariable (docetaxel) and non-linear (paclitaxel) pharmacokinetics,serious hypersensitivity reactions associated with the formulationvehicle (Cremophor E L, Tween 80), and dose-limiting myelosuppressionand neurotoxicity. In the case of Taxotere®, the large variability inpharmacokinetics causes significant variability in toxicity andefficacy, as well as hematological toxicity correlated with systemicexposure to the unbound drug. In addition, since the therapeuticactivity of taxanes increases with the duration of tumor cell drugexposure, the dose-limiting toxicity of commercial taxane formulationssubstantially limits their therapeutic potential.

Accordingly, there is a need in the art for strategies to enable a widevariety of drugs to be formulated as LN and thus realize the benefits ofliposomal delivery technology.

SUMMARY

In one aspect, a drug derivative of formula I is provided:

wherein

-   -   D is a drug;    -   n is 1, 2, or 3; and    -   Z is a Liposome Solubilization Unit of formula II:

-   -   wherein        -   [L] is a Linker selected from the group consisting of:            carboxy, carboxyamido, and alkyl silyl.        -   [S] is a Spacer selected from the group consisting of:            -   C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, each                optionally substituted with one or more substituents                selected from the group consisting of: halo; C₁-C₁₀                alkyl; cycloalkyl; and —YR², wherein                -   Y is a heteroatom selected from the group consisting                    of: N, O, S, and Si, and                -   R² is selected from the group consisting of: H; a                    heteroatom selected from the group consisting of N,                    O, and S; C₁-C₁₀ alkyl; and cycloalkyl, each                    optionally substituted with halo;            -   C₁-C₁₀ heteroalkyl, optionally substituted one or more                times with —YR², wherein                -   Y is a heteroatom selected from the group consisting                    of N, O, S, and Si, and                -   R² is selected from the group consisting of: H; a                    heteroatom selected from the group consisting of: N,                    O, and S; C₁-C₁₀ alkyl; and cycloalkyl, each                    optionally substituted with halo; and            -   cycloalkyl, heterocyclyl, aryl, and heteroaryl, each                optionally substituted with halo; and        -   [N] is a Solubilization Domain of the general formula III:

-   -   -   -   wherein                -   R and R′ are independently selected from the group                    consisting of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,                    C₂-C₁₀ alkynyl, each optionally substituted with                    halo; cycloalkyl, heterocyclyl, aryl, and                    heteroaryl, each optionally substituted with halo;                    and a protonable nitrogen-containing heterocyclic                    system; or                -   R and R′ together with the nitrogen atom to which                    they are attached form a heterocyclic ring having                    four to five carbon atoms, which may comprise one of                    multiple rings within a ring system.

In some aspects, [N] has a pKa of at least about 5.5.

In further aspects, [N] has a pKa less than or equal to about 12.0.

In some aspects, the drug derivative is suitable to be actively loadedinto liposomal nanoparticles having an aqueous interior.

In further aspects, the drug derivative is suitable to be activelyloaded into the aqueous interior of the liposomal nanoparticles. In someaspects, the aqueous interior of the liposomal nanoparticles has anacidic pH relative to the external medium. In further aspects, the drugderivative is protonated within the aqueous interior of the liposomalnanoparticles.

In other aspects, the drug derivative is suitable to be actively loadedso that the drug derivative resides within or is stably associated withthe liposomal nanoparticle membrane. In some of these aspects, [N] isselected from a group of formula IVa or IVb:

wherein:

-   -   A is selected from the group consisting of: carbonyl, methylene,        and NR—C═O, where R is H or C₁-C₅ alkyl;    -   R¹ and R² are independently selected from the group consisting        of: linear or branched C₁-C₃₀ alkyl, C₂-C₃₀ alkenyl, and C₂-C₃₀        alkynyl; and    -   R³ and R⁴ are independently selected from the group consisting        of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, each        optionally substituted with halo; and cycloalkyl, heterocyclyl,        aryl, and heteroaryl, each optionally substituted with halo; or    -   R³ and R⁴ together with the nitrogen atom to which they are        attached form a heterocyclic ring having four to five carbon        atoms, which may comprise one of multiple rings within a ring        system.

Also provided herein is a liposomal nanoparticle formulation of a drugderivative provided herein. In some aspects, the liposomal nanoparticleformulation is formed by actively loading the drug derivative intoliposomal nanoparticles having an aqueous interior.

In further aspects, the drug derivative resides within the aqueousinterior of the liposomal nanoparticles. In some aspects, the aqueousinterior of the liposomal nanoparticles has an acidic pH relative to theexternal medium. In further aspects, the drug derivative is protonatedwithin the aqueous interior of the liposomal nanoparticles.

In yet further aspects, the drug derivative resides within or is stablyassociated with the liposomal nanoparticle membrane. In further aspects,[N] is a group of formula IVa or IVb:

wherein:

-   -   A is selected from the group consisting of: carbonyl, methylene,        and NR—C═O, where R is H or C₁-C₅ alkyl;    -   R¹ and R² are independently selected from the group consisting        of: linear or branched C₁-C₃₀ alkyl, C₂-C₃₀ alkenyl, and C₂-C₃₀        alkynyl; and    -   R³ and R⁴ are independently selected from the group consisting        of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, each        optionally substituted with halo; and cycloalkyl, heterocyclyl,        aryl, and heteroaryl, each optionally substituted with halo; or    -   R³ and R⁴ together with the nitrogen atom to which they are        attached form a heterocyclic ring having four to five carbon        atoms, which may comprise one of multiple rings within a ring        system.

In another aspect, a pharmaceutical composition is provided hereincomprising a liposomal nanoparticle formulation of a drug derivativeprovided herein and a pharmaceutically acceptable excipient.

In an additional aspect, a method of modifying a drug to facilitateloading of the drug into LN is provided herein, the method comprisingconjugating a Liposome Solubilization Unit (Z) of formula II to the drug

wherein

-   -   [L] is a Linker selected from the group consisting of: carboxy,        carboxyamido, and alkyl silyl.    -   [S] is a Spacer selected from the group consisting of:        -   C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, each            optionally substituted with one or more substituents            selected from the group consisting of: halo; C₁-C₁₀ alkyl;            cycloalkyl; and —YR², wherein            -   Y is a heteroatom selected from the group consisting of:                N, O, S, and Si, and            -   R² is selected from the group consisting of: H; a                heteroatom selected from the group consisting of N, O,                and S; C₁-C₁₀ alkyl; and cycloalkyl, each optionally                substituted with halo;        -   C₁-C₁₀ heteroalkyl, optionally substituted one or more times            with —YR², wherein            -   Y is a heteroatom selected from the group consisting of                N, O, S, and Si, and            -   R² is selected from the group consisting of: H; a                heteroatom selected from the group consisting of: N, O,                and S; C₁-C₁₀ alkyl; and cycloalkyl, each optionally                substituted with halo; and        -   cycloalkyl, heterocyclyl, aryl, and heteroaryl, each            optionally substituted with halo; and    -   [N] is a Solubilization Domain of the general formula III:

-   -   -   wherein            -   R and R′ are independently selected from the group                consisting of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀                alkynyl, each optionally substituted with halo;                cycloalkyl, heterocyclyl, aryl, and heteroaryl, each                optionally substituted with halo; and a protonable                nitrogen-containing heterocyclic system; or            -   R and R′ together with the nitrogen atom to which they                are attached form a heterocyclic ring having four to                five carbon atoms, which may comprise one of multiple                rings within a ring system.

In still additional aspects, a method of loading a drug into liposomalnanoparticles is provided herein, the method comprising the steps ofconjugating a Liposome Solubilization Unit (Z) of formula II to the drugto form a drug derivative; and actively loading the drug derivative intoliposomal nanoparticles having an aqueous interior

wherein

-   -   formula II is:

-   -   [L] is a Linker selected from the group consisting of: carboxy,        carboxyamido, and alkyl silyl.    -   [S] is a Spacer selected from the group consisting of:        -   C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, each            optionally substituted with one or more substituents            selected from the group consisting of: halo; C₁-C₁₀ alkyl;            cycloalkyl; and —YR², wherein            -   Y is a heteroatom selected from the group consisting of:                N, O, S, and Si, and            -   R² is selected from the group consisting of: H; a                heteroatom selected from the group consisting of N, O,                and S; C₁-C₁₀ alkyl; and cycloalkyl, each optionally                substituted with halo;        -   C₁-C₁₀ heteroalkyl, optionally substituted one or more times            with —YR², wherein            -   Y is a heteroatom selected from the group consisting of                N, O, S, and Si, and            -   R² is selected from the group consisting of: H; a                heteroatom selected from the group consisting of: N, O,                and S; C₁-C₁₀ alkyl; and cycloalkyl, each optionally                substituted with halo; and        -   cycloalkyl, heterocyclyl, aryl, and heteroaryl, each            optionally substituted with halo; and    -   [N] is a Solubilization Domain of the general formula III:

-   -   -   wherein            -   R and R′ are independently selected from the group                consisting of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀                alkynyl, each optionally substituted with halo;                cycloalkyl, heterocyclyl, aryl, and heteroaryl, each                optionally substituted with halo; and a protonable                nitrogen-containing heterocyclic system; or            -   R and R′ together with the nitrogen atom to which they                are attached form a heterocyclic ring having four to                five carbon atoms, which may comprise one of multiple                rings within a ring system.

In some aspects, the drug derivative is actively loaded into the aqueousinterior of the liposomal nanoparticles. In further aspects, the aqueousinterior of the liposomal nanoparticles has an acidic pH relative to theexternal medium. In yet further aspects, the drug derivative isprotonated within the aqueous interior of the liposomal nanoparticles.

In some aspects, the drug derivative is actively loaded so that itresides within or is stably associated with the liposomal nanoparticlemembrane. In further aspects, [N] is a group of formula IVa or IVb:

wherein:

-   -   A is selected from the group consisting of: carbonyl, methylene,        and NR—C═O, where R is H or C₁-C₅ alkyl;    -   R¹ and R² are independently selected from the group consisting        of: linear or branched C₁-C₃₀ alkyl, C₂-C₃₀ alkenyl, and C₂-C₃₀        alkynyl; and    -   R³ and R⁴ are independently selected from the group consisting        of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, each        optionally substituted with halo; and cycloalkyl, heterocyclyl,        aryl, and heteroaryl, each optionally substituted with halo; or    -   R³ and R⁴ together with the nitrogen atom to which they are        attached form a heterocyclic ring having four to five carbon        atoms, which may comprise one of multiple rings within a ring        system.

In yet another aspect, a method is provided for treating a disease orcondition, comprising administering an effective amount of apharmaceutical composition described herein to a patient in need oftreatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Kinetics of prodrug hydrolysis in pH 7.4 aqueous buffer andphosphate-buffered mouse plasma (pH 7.4) at 37° C.

FIG. 2. Loading of docetaxel derivatives into LN. The derivatives wereloaded by incubation at 60° C. into DSPC/chol LN through a pH (ammoniumion) gradient formed by 300 mM ammonium sulfate within LN and anexternal ammonium sulfate-free medium buffered at pH 5. (A) Loadingefficiency of the docetaxel prodrug TD1 at prodrug-to-lipid ratios of0.1 wt/wt (▪), 0.2 wt/wt (♦) and 0.4 wt/wt (▴). (B) Loading efficiencyof C-2′-piperazinyl ester (TD1-TD3), C-2′-piperidine ester (TD7), andC-7-amino ester (TD10) derivatives of docetaxel incubated with DSPC/CholLN at a prodrug-to-lipid ratio of 0.2 wt/wt.

FIG. 3. Loading of prednisone derivatives. into LN. The derivatives wereloaded at 60° C. into DSPC/Chol LN through a pH (ammonium ion) gradientformed by 300 mM ammonium sulfate within LN and an external ammoniumsulfate-free medium buffered at pH 5. (A): Loading efficiency of anN-methyl-piperazinyl butanoic acid ester derivative of prednisone (♦)relative to the parent drug (▪) at a drug-to-lipid ratio of 0.12 wt/wt.The prednisone derivative did not spontaneously partition into the LNbilayer; thus, there was no measurable amount of the derivativeassociated with the LN carrier in the absence of the ammonium sulfate(pH) gradient. (B): Loading efficiencies of prednisone derivatives withvarying linker lengths after 15 min of incubation at 60° C.: comparisonof N-methyl-piperazinyl butanoic acid ester (B) and N-methyl-piperazinylacetic acid ester (E) derivatives. The N-methyl-piperazinyl butanoicacid ester derivative showed 100% loading efficiency whereas theN-methyl-piperazinyl acetic acid ester derivative showed about 75%loading efficiency.

FIG. 4. Loading of an etoposide derivative into LN. TheN-methyl-piperazinyl butanoic acid ester derivative was loaded at 60° C.into DMPC/Chol LN through a pH (ammonium ion) gradient formed by 300 mMammonium sulfate within LN and an external ammonium sulfate-free mediumbuffered at pH 5. The derivative showed 100% loading efficiency within15 min of incubation at 60° C. at a drug-to-lipid ratio of 0.16 wt/wt.

FIG. 5. Formulation stability. The stability of LN-docetaxel derivativeformulations with different lipid compositions (DSPC/Chol, DPPC/Chol andDMPC/Chol) was followed over a period of 4 months upon cold storage (7°C.). The prodrug-to-lipid ratios of the formulations were 0.2 wt/wt. (A)Prodrug hydrolysis; increase of parent drug (docetaxel) was determinedby UHPLC, (B) percentage of the prodrug retained in LN, (C) LN size andpolydispersity measured by dynamic light scattering.

FIG. 6. Plasma elimination profiles of Taxotere™ (▴), TD1 formulated inthe same manner as Taxotere™ (ethanol/polysorbate 80/physiologicalsaline) (▪) and DSPC/Chol LN formulation of TD1 (prodrug-to-lipid ratio0.2 (wt/wt)) (♦) following i.v. administration in mice. Female SwissWebster mice were injected intravenously with a single dose of thevarious formulations at equimolar docetaxel doses (20 mg/kg docetaxel).Prodrug levels in plasma were determined by UHPLC-MS. Data pointsrepresent mean values±standard deviation from each group of mice (n=4).

FIG. 7. Plasma drug retention profiles. Retention of the docetaxelderivative TD1 in DSPC/Chol (♦), DPPC/Chol (▪) and DMPC/Chol (▴) LNformulations was determined in vitro (A) and in vivo (B). In vitroretention of TD1 in DSPC/chol LN was compared with other docetaxelderivatives (TD2-3 and TD7) formulated in DSPC/chol LN at the samedrug-to-lipid ratio in mouse plasma (C). LN formulations containingtrace amounts of the radiolabeled lipid [³H]-CHE were injectedintravenously into female Swiss Webster mice at a docetaxel equivalentdose of 20 mg/kg or incubated in vitro at 37° C. in mouse plasma. Plasmasamples taken at the indicated time points were analyzed for lipid andprodrug content by liquid scintillation counting and UHPLC,respectively. For the in vitro retention studies unentrapped (released)drug was removed from the plasma samples by size exclusionchromatography using Sephadex G50 spin columns prior to analysis oflipid and drug content. Data points represent means±standard deviations(n=4).

FIG. 8. Anticancer efficacy. Response of subcutaneous MDA435/LCC6 humanbreast carcinoma xenografts to treatment with Taxotere™ andLN-encapsulated TD1 in Rag2M mice. (A) Treatment with various LNformulations to determine the effect of lipid composition on efficacy.LN formulations (prodrug-to-lipid ratio 0.2 wt/wt) were composed ofDSPC/Chol (▪), DPPC/Chol (▴) and DMPC/Chol (*) and administered at adocetaxel equivalent dose of 40 mg/kg. Untreated control received asaline injection (♦). (B) Dose-response for the DSPC/Chol LN formulation(prodrug-to-lipid ratio 0.2 wt/wt) administered at docetaxel equivalentdoses of 25 (x), 40 (*) and 88 (▴) mg/kg. Untreated controls received asaline injection (♦). Taxotere™ at 25 mg/kg docetaxel was included forcomparison (▪). (C) Comparison of Taxotere™ with the 88 mg/kg DSPC/CholLN formulation. Tumor growth curves are shown with standard deviations.Treatment was initiated at day 35 with a single i.v. bolus injection.Points represent the means of relative tumor volumes (ratio of the tumorvolume measured at a given time point to the tumor volume measured atthe treatment day); mean values for 6 mice per group are presented.

FIG. 9. In vivo kinetics and anticancer activity of free vincristine(VCR) (2 mg/kg) and vincristine (2 mg/kg) encapsulated in 100 nm eggsphingomyelin/cholesterol LN injected i.v. in SCID mice bearing A431tumors. (8A) Concentration profile of free VCR (□) and LN formulated VCR() in blood plasma over time. Free VCR is rapidly removed fromcirculation, whereas LN formulated VCR has an extended circulationhalf-life. (8B) Release of free VCR () from LN formulations (%retention) over time. LN formulated VCR exhibits a sustained releaseprofile in circulation. (8C) Tumor concentration (μg/ml) of VCR overtime after administration of free VCR (□) and LN formulated VCR (). Theextended half-life and sustained release profile of LN formulated VCRresults in increasing tumor accumulation of VCR over time. (8D)Anticancer activity of free VCR (□) and LN formulated VCR () relativeto saline control (▪). LN formulated VCR has significantly greateranticancer activity than the free compound. Nano-liposomal formulationsincrease the amount of drug reaching sites of tumor growth and prolongsthe duration of exposure to therapeutically active levels of drug,resulting in increased antitumor activity.

FIG. 10. Schematic illustration of the chemistry strategy employed forthe synthesis of weak base drug derivatives based on esterification ofhydroxyl groups located on the drug.

DETAILED DESCRIPTION

As used herein, the term “liposome” or “liposomal nanoparticle” or “LN”refers to a self-assembling structure comprising one or more lipidbilayers, each of which comprises two monolayers containing oppositelyoriented amphipathic lipid molecules. Amphipathic lipids comprise apolar (hydrophilic) headgroup covalently linked to one or two or morenon-polar (hydrophobic) acyl or alkyl chains. Energetically unfavorablecontacts between the hydrophobic acyl chains and a surrounding aqueousmedium induce amphipathic lipid molecules to arrange themselves suchthat polar headgroups are oriented towards the bilayer's surface andacyl chains are oriented towards the interior of the bilayer,effectively shielding the acyl chains from contact with the aqueousenvironment.

Liposomes useful in connection with the methods and compositionsdescribed herein can have a single lipid bilayer (unilamellar liposomes)or multiple lipid bilayers (multilamellar liposomes) surrounding orencapsulating an aqueous compartment. Various types of liposomes aredescribed, e.g., in Cullis et al., Biochim. Biophys Acta, 559: 399-420(1987).

Amphipathic lipids typically comprise the primary structural element ofliposomal lipid vesicles. Hydrophilic characteristics of lipids derivefrom the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl,nitro, and other like polar groups. Hydrophobicity can be conferred bythe inclusion of groups that include, but are not limited to, long chainsaturated and unsaturated aliphatic hydrocarbon groups, which may besubstituted by one or more aromatic, cycloaliphatic or heterocyclicgroup(s). Examples of preferred amphipathic compounds arephosphoglycerides and sphingolipids, representative examples of whichinclude phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,phoasphatidylglycerol, palmitoyloleoyl phosphatidylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylcholine, distearoylphosphatidylcholine(DSPC), dilinoleoylphosphatidylcholine and egg sphingomyelin. Otherlipids such as sphingolipids and glycosphingolipids, are also useful inmethods and compositions provided herein. Additionally, the amphipathiclipids described above may be mixed with other lipids, such astriacylglycerols and sterols.

As used herein, the term “C₁₋₁₀-alkyl” refers to a linear or branchedsaturated hydrocarbon chain wherein the longest chain has from one toten carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl,etc.

As used herein, the term “C₂₋₁₀-alkenyl” means a linear or branchedhydrocarbon group having from two to ten carbon atoms and containing oneor more double bonds. Non-limiting examples of C₂₋₁₀-alkenyl groupsinclude allyl, homo-allyl, vinyl, crotyl, butenyl, pentenyl, hexenyl,heptenyl, octenyl, etc. Non-limiting examples of C₂₋₁₀-alkenyl groupswith more than one double bond include butadienyl, pentadienyl,hexadienyl, heptadienyl, heptathenyl, octatrienyl, etc. groups as wellas branched forms of these. The position of the unsaturation (the doublebond) may be at any position along the carbon chain.

As used herein, the term “C₂₋₁₀-alkynyl” refers a linear or branchedhydrocarbon group containing from two to eight carbon atoms andcontaining one or more triple bonds. Non-limiting examples ofC₂₋₁₀-alkynyl groups include ethynyl, propynyl, butynyl, pentynyl,hexynyl, heptynyl, octynyl, etc. groups as well as branched forms ofthese. The position of unsaturation (the triple bond) may be at anyposition along the carbon chain. More than one bond may be unsaturatedsuch that the “C₂₋₁₀-alkynyl” is a di-yne or enedi-yne.

As used herein, the term “heteroalkyl” indicates an alkane groupcontaining 1 or more, and preferably 1 or 2, heteroatoms selected fromO, S and N. Where present, such heteroatoms are optionally furthersubstituted by a heteroatom selected from O, S, N, and Si, or an alkyl,alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl group optionallysubstituted with halo. Non-limiting examples include (one or more)ether, thioether, ester and amide groups.

As used herein, the terms “aryl” and “cycloalkyl” refer to mono- andbicyclic ring structures comprising 5 to 12 carbon atoms, and preferablyto monocyclic rings comprising 5 to 6 carbon atoms. Where such ringscomprise one or more heteroatoms, selected from N, S and O, (i.e.,heterocyclic, or heteroaryl rings) such rings comprise a total of 5 to12 atoms, more preferably 5 to 6 atoms. Heterocyclic rings include, butare not limited to, furyl, pyrrolyl, pyrazolyl, thienyl, imidazolyl,indolyl, benzofuranyl, benzothiophenyl, indazolyl, benzoimidazolyl,benzothiazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, pyridyl,piperidinyl, piperazinyl, pyridazinyl, pyrimidinyl, pyrazinyl,morpholinyl, oxadiazolyl, thiadiazolyl, imidazolinyl, imidazolidinyl andthe like. The ring may be substituted with one or more heteroatomsselected from O, S, and N. Where present, such heteroatoms areoptionally further substituted by a heteroatom selected from O, S, N,and Si, or an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroarylgroup optionally substituted with halo.

The substituents C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₁₋₁₀alkoxy, C₁₋₁₀ heteroalkyl, C₁₋₁₀ aminoalkyl, C₁₋₁₀ haloalkyl and/orC₁₋₁₀ alkoxycarbonyl may, if present, be substituted by one or more ofhydroxyl, C₁₋₆ alkoxy, halogen, cyano, amino or nitro.

As used herein, the term “halogen” or “halo” includes chlorine,fluorine, which are preferred, and iodine and bromine.

The present invention relates generally to a medicinal chemistryplatform for modifying drugs to facilitate loading of the drugs intoliposomal nanoparticles (LN). In some preferred aspects,lipophilic/water-insoluble drugs that are resistant or incapable ofbeing encapsulated into liposomes using standard techniques are modifiedto form drug derivatives that can be efficiently loaded into LNexhibiting a pH gradient across the liposomal membrane. While variousaspects of the invention are described in relation to chemotherapeuticagents, methods and compositions provided herein represent a flexibletechnology platform that can be used with any drug or therapeutic agentfor which liposomal delivery would be beneficial, including but notlimited to, established chemotherapeutic agents and drugs for treatingcancers, inflammatory conditions, infectious diseases, and otherindications.

Also provided herein are drug derivatives capable of being efficientlyloaded into LN exhibiting a transmembrane pH or ion gradient, as well asLN formulations and pharmaceutical compositions comprising such drugderivatives. In various embodiments, the drug derivatives are preparedby chemically modifying known drugs having one or more properties, suchas but not limited to, poor aqueous solubility, that prevent them frombeing efficiently loaded into liposomes. In some preferred embodiments,the drug is a chemotherapeutic agent.

In some aspects, drug derivatives provided herein are formed byderivatizing a drug with a “solubilizing unit” which possesses one ormore characteristics that facilitate loading of the derivatized druginto LN. In various aspects, the solubilizing unit is physiochemicallytailored to facilitate efficient loading of a derivatized drug into LNand/or efficient release of the drug from the LN under preferredconditions at or near a therapeutic target.

In some preferred aspects, the solubilizing unit comprises a weaklybasic amino group which facilitates active loading of the drugderivative into LN in the presence of a transmembrane pH or iongradient. As used herein, the term “weak base derivative” refers to adrug modified according to methods provided herein to contain a weaklybasic moiety, such as a primary, secondary or tertiary amine.

In some aspects, the weak base moiety is an ionizable amino group, suchas an N-methyl-piperazino group, a morpholino group, a piperidino group,a bis-piperidino group or a dimethylamino group. Examples of modifyinggroups for the synthesis of weak base drug derivatives include, but arenot limited to, N-methyl-piperazino (e.g., as in the anticancer drugGlivec®), bis-piperazino, bis-piperidino (e.g., as in the anticancerdrug irinotecan), piperidino, morpholino, dimethylamino, aminomethyl(glycine), aminoethyl (alanine), and aminobutyryl groups, and lipidswith a protonable amine group.

In some aspects, the weakly basic amino group is selected from the groupconsisting of:

where n is between 1 and about 10, or more preferably 1 and 4.

In some aspects, the solubilization unit further comprises a linker unitwhich facilitates attachment of the solubilization unit to the drugtargeted for derivatization. In some aspects, the linker comprises areactive carbonyl group (e.g., a carboxylic acid moiety) which reactswith a free OH group on the drug to form a carboxylester linkage. Infurther aspects, the linker comprises a dialkylsilyl group which reactswith a free OH group on the drug to form a silyl ether linkage. In otheraspects, the linker comprises a carbamate group.

In various aspects, drug derivatives of the following general structureare provided, where Z is a water-solubilizing unit, D is a drug, and nis 1, 2, or 3:

In some aspects, Z comprises a group of formula II, wherein:

the wavy line represents the bond connecting Formula IIA to a reactivegroup, such as a free O atom, in the drug;

[L] is a Linker selected from the group consisting of: carboxy,carboxyamido, and alkyl silyl;

[S] is a Spacer selected from the group consisting of:

-   -   C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, and C₂-C₁₀ alkynyl, each        optionally substituted with one or more substituents selected        from the group consisting of: halo; C₁-C₁₀ alkyl; cycloalkyl;        and —YR², wherein        -   Y is a heteroatom selected from the group consisting of: N,            O, S, and Si, and        -   R² is selected from the group consisting of: H; a heteroatom            selected from the group consisting of N, O, and S; C₁-C₁₀            alkyl; and cycloalkyl, each optionally substituted with            halo;    -   C₁-C₁₀ heteroalkyl, optionally substituted one or more times        with —YR², wherein        -   Y is a heteroatom selected from the group consisting of N,            O, S, and Si, and        -   R² is selected from the group consisting of: H; a heteroatom            selected from the group consisting of: N, O, and S; C₁-C₁₀            alkyl; and cycloalkyl, each optionally substituted with            halo; and    -   cycloalkyl, heterocyclyl, aryl, and heteroaryl, each optionally        substituted with halo; and

[N] is a Solubilization Domain, comprising a weakly basic group thatfacilitates loading of the drug derivative into LN exhibiting atransmembrane pH or ion gradient, where [N] is of the general formulaIII, wherein:

-   -   R and R′ are independently selected from the group consisting        of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, each        optionally substituted with halo; cycloalkyl, heterocyclyl,        aryl, and heteroaryl, each optionally substituted with halo; and        a protonable nitrogen-containing heterocyclic system; or    -   R and R′ together with the nitrogen atom to which they are        attached form a heterocyclic ring having four to five carbon        atoms, which may comprise one of multiple rings within a ring        system.

In various aspects, the solubilizing unit is tailored to facilitateactive loading of the drug derivative to specific locations within theLN. For example, in some aspects, a drug is derivatized with awater-solubilizing unit that facilitates active loading of the drugderivative into the aqueous interior of the LN. The addition of an aminegroup is a common drug modification strategy to improve water-solubility(e.g., Capdeville et al., Nature Reviews Drug Discovery, 1: 493-502(2002); Pizzolato and Saltz, Lancet, 361: 2235-2242 (2003)), and avariety of methods are known in the art for making amine drugderivatives, including reversible drug conjugates (e.g., groups removedin vivo by enzyme action). Non-limiting examples of amine-modified drugswith improved aqueous solubility include the anticancer agents Glivec(N-methyl-piperazine), irinotecan (bis-piperidine) and topotecan(ethyldimethylamino group).

In other aspects, a drug is derivatized with a lipid-solubilizing unitto facilitate active loading of the drug derivative such that thederivative resides in, or is stably associated with, the liposomalmembrane. In some aspects, the lipid-solubilizing unit comprises aweakly basic group and a lipophilic group. The lipophilic group may beselected to facilitate active loading of the drug into LN, stability ofthe drug within LN, and/or the release of the drug at or near atherapeutic target. In some aspects, the lipophilic group has a similaror complementary lipid composition as the liposomal membrane. In somesuch aspects, the lipid-solubilizing unit is selected from a group offormula IVa or IVb:

wherein:

-   -   A is selected from the group consisting of: carbonyl, methylene,        and NR—C═O, where R is H or C₁-C₅ alkyl;    -   R¹ and R² are independently selected from the group consisting        of: linear or branched C₁-C₃₀ alkyl, C₂-C₃₀ alkenyl, and C₂-C₃₀        alkynyl; and    -   R³ and R⁴ are independently selected from the group consisting        of: H; C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, each        optionally substituted with halo; and cycloalkyl, heterocyclyl,        aryl, and heteroaryl, each optionally substituted with halo; or    -   R³ and R⁴ together with the nitrogen atom to which they are        attached form a heterocyclic ring having four to five carbon        atoms, which may comprise one of multiple rings within a ring        system.

Weak base derivatives can be loaded into LN by imposing a pH gradientacross the liposome membrane (inside acidic) and incubating the liposomewith the drug to be encapsulated. Depending on the pH, weak basederivatives can exist in either a charged (protonated) form (e.g., wherethe pH is below the pKa) or a neutral form (e.g., where the pH is at orabove the pKa). Only the neutral form can rapidly permeate across theliposome membrane. Upon reaching the acidic liposome interior, thecharged membrane-impermeable form is adopted, driving the continueduptake and retention of the compound in the liposome interior.

In some preferred aspects, the drug loading properties of a weak basederivative provided herein can be fine-tuned by selecting and/ormodifying one or more properties of derivative amine groups and/orderivative lipophilic groups. For example, the pKa of a derivative aminegroup can be selected such that the amine group is protonated in theaqueous interior of the LN preparation being used (e.g., at low pH) andunprotonated in the external medium (e.g., at neutral or basic pH).

In some aspects, the pKa of a derivative amine group is less than orequal to about 12.0, less than or equal to about 11.5, less than orequal to about 11.0, less than or equal to about 10.5, less than orequal to about 10.0, less than or equal to about 9.5, or less than orequal to about 9.0.

In some aspects, the pKa of a derivative amine group is at least about5.0, at least about 5.5, at least about 6.0, at least about 6.5, atleast about 7.0, at least about 7.5, at least about 8.0, or at leastabout 8.5.

A solubilizing unit comprising a weak base moiety can be attached to anysuitably reactive functional group on the drug targeted formodification. Such functional groups include hydroxyl, sulfhydryl andcarboxyl groups among others. In some aspects, the solubilizing unit isattached via a free OH group on the drug, for example, by acarboxylester bond.

In some aspects, drugs are derivatized in a region that is not essentialfor the intended therapeutic activity such that the activity of thederivative is substantially equivalent to that of the free drug. Forexample, in some aspects, the weak base derivative comprises the taxanedocetaxel derivatized at the 7-OH group of the baccatin skeleton.

In further aspects, drugs are derivatized in a region that is essentialfor activity such that the derivatives are prodrugs that must beconverted to the parent compound or another active form in order toexert the intended therapeutic effect. For example, in some aspects,docetaxel derivatives are provided herein which are derivatized at the2′-OH group which is essential for docetaxel activity.

Prodrug derivatives provided herein are preferably rapidly converted tothe free drug upon release from the LN carrier and exposure tophysiological conditions in vivo. For example, in some preferredaspects, the derivative amino group of a weak base derivative providedherein is removed rapidly from the drug following release from theliposome, for example via the action of endogenous enzymes and/or byspontaneous hydrolysis.

Thus, in some aspects, the solubilizing unit is reversibly conjugated tothe drug derivative to form a prodrug which is stable under certainconditions (e.g., during loading, formulation, storage, and/oradministration of a LN composition) and dissociates to release the freedrug at or near its therapeutic target(s), for example by the action ofendogenous enzymes and/or under certain physiological conditions (e.g.,pH, ionic strength).

In some aspects, weak base derivatives are engineered to be stableinside the liposomal nanocarrier (e.g., at low pH) but are‘self-releasing’ at physiological pH, such that the prodrug is rapidlyconverted into its active form upon release from the liposome. This canbe achieved by, e.g., attaching an aminobutyryl group to docetaxel,which in its unprotonated form (pH 7.4) can trigger release throughnucleophilic attack on the ester carbonyl.

In some aspects, the hydrolytic stability of an ester linkage of a weakbase derivative may be modulated by exploiting one or more of thefollowing effects:

i) inductive effects: Esters may be stabilized or destabilized towardcleavage at physiological pH, either with assistance of proteases or byspontaneous hydrolysis, or by positioning the solubilizing amino groupcloser to (destabilization) or farther away from (stabilization) thecarbonyl center. The pKa of the amino group also plays a role in thiscontext: a charged (protonated) amine promotes ester hydrolysis underphysiological conditions. By appropriate choice of groups R groups onthe amino group the N-center can be modulated to achieve an ideal rateof ester cleavage.

ii) Chemical and proximity effects. Esterification with, e.g., a4-aminobutyrl group in which the amino unit has a pKa˜6 produces anentity which will exist in its protonated form at low pH, such as the pHfound inside ammonium sulfate-loaded LN. Release of such derivativesfrom the LN carrier and exposure to physiological conditions (e.g., pH7.4) promotes formation of a free base form, allowing the free amine totrigger “self-release” of the derivatizing unit through nucleophilicattack on the ester carbonyl. Advantageously, this allows prodrugderivatives to be rapidly converted into active form upon release fromLN.

In further aspects, the hydrolytic stability of a silyl ether linkage ofa weak base derivative may be modulated to facilitate spontaneoushydrolysis. Unlike with ester linkers, there are no endogenousdesilylating enzymes. Thus, derivatives comprising a silyl ether linkagepreferably comprise a linker group that allows for hydrolysis underphysiological conditions. The primary determinant of the rate of silylgroup cleavage is the steric bulk around the Si atom. Modulation of thisphysiochemical property entails varying the size of groups R and R′ insilyl halides, e.g., through the series Me, Et, i-Pr, t-Bu, Ph, etc. Asin the case of the esters, the pKa of the amino group also plays a rolein defining the stability of derivatives. For example, an amino groupwith a pKa˜6 will exist predominantly as the free base at physiologicalpH, thereby facilitating the cleavage of the silyl group.

In further aspects, the size and/or chemical composition of a lipophilicderivative group can be selected to enhance solubility in the liposomalmembrane and/or stability of the drug within the liposome (e.g., byanchoring the drug in the liposomal membrane).

Advantageously, weak base derivatives of a drug provided herein can beloaded into liposomes more efficiently than the free drug. In someaspects, a weak base derivative provided herein can be loaded intoliposomes with a loading efficiency or at least about 50%, at leastabout 55%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, or higher over a wide range ofdrug-to-lipid ratios (e.g., from about 0.01 mg/mg, to about 10 mg/mg orhigher).

In further aspects, LN formulations of weak base derivatives providedherein can be optimized to achieve sustained release of the drugderivative through, e.g., modification of the lipid composition of theLN carrier membrane. For example, in various aspects, LN formulationsprovided herein have a release half-life in vivo of between about 1 toabout 96 hours, or about 2 to about 72 hours, or about 4 to about 48hours.

Methods provided herein can be used to modify any drug or therapeuticagent for which a liposomal formulation is desirable. In some preferredaspects, the drug is lipophilic and/or poorly water-soluble.Advantageously, modification of such drugs according to methods providedherein results in improved solubility, reduced toxicity, increasedefficacy, and/or other benefits relative to the free drug.

Non-limiting examples of drugs that can be modified and loaded into LNaccording to methods provided herein are given in Table 1.

TABLE 1 Exemplary drugs for derivatization. Solubility in Water DrugIndication (μg/ml) Amprenavir HIV 36 (49 pred.) BexaroteneAntineoplastic 0.15 (pred.) Calcitrol Calcium regulator 6.7 (pred.)Cyclosporin A Immunosuppressant 9.5 (pred.) Digoxin Heart failure 127(pred.) Doxercalciferol Hyperparathyroism relatively insolubleParicalcitol Hyperparathyroism 6.8 Dronabinol Anorexia 2800 (pred.)insoluble in water, oil at room temp. Etoposide Antineoplastic 58.7, 200Teniposide Antineoplastic 59.8 (pred.) Isotretinoin Antiacne 4.8 (pred.)Sirolimus Antineoplastic 1.7 (pred.), insoluble in water TretinoinAntineoplastic 1000, 4.7 (pred.) Valproic acid Antiepileptic 1300Amphotericin B Antifungal 750 Docetaxel Antineoplastic 12.7 (pred.)Paclitaxel Antineoplastic 5.5 (pred.) Fulvestrant Antineoplastic 6.7(pred.) Tacrolimus Immunosuppressant 4 (pred.), insoluble ValrubicinAntineoplastic 32.5 (pred.), insoluble Propofol Anesthetic 124Prednisone Anti-inflammatory 312 Prednisolone Anti-inflammatory 223Dexamethasone Anti-inflammatory 89 Tacrolimus (FK- Immunosuppressive 4(pred.), insoluble 506) Mycophenolic Immunosuppressive, anti- 35.5(pred.), insoluble acid proliferative Lovastation Anti-cholesteremic 24(pred.) pred. = predicted Sources: R. G. Strickley, Pharm. Res. 21(2):201 (2004); DrugBank at http://www.drugbank.ca/

In some preferred aspects, the drug is a chemotherapeutic agent.Examples of established drugs or classes of drugs that can bederivatized according to methods provided herein include the taxanes(e.g. paclitaxel and docetaxel) and the podophyllotoxin derivatives(e.g. etoposide and teniposide). The taxanes, which include docetaxel(Taxotere) and paclitaxel (Taxol), are an important family of drugs thathave extensive use in clinical oncology. Like most anticancer drugs,taxanes are non-selective for cancer cells and can also cause damage tohealthy cells. Because taxanes are poorly soluble in aqueous solution,they are typically formulated in vehicles such as Cremophor andPolysorbate 80, which themselves cause adverse reactions in patients.Steroidal and anti-allergy pre-medication is often used to minimize theside effects of the vehicle. Advantageously, LN taxane formulationsprovided herein allow for administration of taxanes without use of atoxic vehicle. Moreover, because LN can exit the bloodstream andpreferentially accumulate at high concentrations in tumors due to the“leaky” nature of blood vessels at these sites, LN taxane formulationscan offer superior anti-cancer activity with fewer side effects (e.g.,improved therapeutic index) compared to Taxotere, the approvedformulation of the parent compound.

In some aspects, LN formulations provided herein increase the amount ofa chemotherapeutic agent that specifically reaches a site of tumorgrowth and/or prolongs the duration of exposure of a tumor totherapeutically active levels of drug, for example through extendedplasma half-life of the LN and/or sustained release of thechemotherapeutic agent from the LN carrier.

In some aspects, the drug is docetaxel (Taxotere®) or paclitaxel, thestructures of which are shown below. The two drugs differ at the levelof the acyl group present on the nitrogen atom of the side chain(tert-butoxycarbonyl, or BOC, in docetaxel; benzoyl in paclitaxel) andin that the C-10 OH group is free in docetaxel, but it is acetylated inpaclitaxel.

Modification of docetaxel and paclitaxel according to methods providedherein involves derivatization of one or more of the free OH units withappropriate groups. In some aspects, the drug is derivatized at the C-1OH. In other, preferred aspects, the drug is derivatized at the C-2′,C-7, and/or C-10 OH to produce the following derivatives, wherein groupsZ connected to the C-2′, C7, and/or C-10 OH are, independently, H, or aresidue containing a protonatable nitrogen functionality. Any drug maybe derivatized in a similar manner as docetaxel and paclitaxel at a freeOH group or other reactive functionality (which can be present on thenative drug or on a modified version of the drug).

In some aspects, LN formulations provided herein have one or morepharmacological properties of liposomal vincristine (Marqibo®), which iscurrently in Phase III clinical trials for the treatment of variouscancers (e.g., Boman et al., Brit. J. Cancer 72: 896-904 (1995), Shan etal., Cancer Chemother. Pharmacol. 58(2): 245-55 (2006), Waterhouse etal., Methods Enzymol. 391: 40-57 (2005)).

In various aspects, drug derivatives of the following general structureare provided, where Z is a water-solubilizing unit and D is a drug:

In some aspects, Z comprises a group of formula IIA, wherein:

(i) the wavy line represents the bond connecting Formula IIA to areactive group, such as a free O atom, in the drug (e.g., compounds 3and/or 4, above);

(ii) [S] is a “spacer” comprising:

(a) a chain of the type (CH₂)_(n), where n may range from 1 to 10, OR

(b) a derivative of the above (CH₂)_(n) where one or more H atoms arereplaced by: a linear, branched, or cyclic alkyl group containing from 1to 10 C atoms, a heteroatom such as N, O, S, Si, which may be furtherconnected to H atoms; or to heteroatoms such as N, O, S; or to linear,branched, or cyclic alkyl groups containing from 1 to 10 C atoms andfacultatively incorporating one or more halogen atoms, a halogen atom;OR

(c) a derivative of the above (CH₂)_(n) where one or more CH₂'s arereplaced by: a heteroatom such as N, O, S, Si, which may be furtherconnected to H atoms; or to heteroatoms such as N, O, S; or to linear,branched, or cyclic alkyl groups containing from 1 to 10 C atoms andfacultatively incorporating one or more halogen atoms, a ring structureconsisting of 3 to 10 carbon atoms and facultatively incorporating oneof more heteroatoms such as N, O, S, Si, or halogen, as well as multiplebonds among pairs of atoms; OR

(d) a derivative of the above (CH₂)_(n) where one or more pairs ofadjacent C atoms share a double bond of E- or Z-geometry, or a triplebond.

Examples of such spacers [S] include, but are not limited to:

(iii) [N] is a Solubilizing Domain, comprising a weakly basic group thatfacilitates loading of the drug derivative into LN exhibiting atransmembrane pH or ion gradient, selected from:

(a) a substituent of general structure R—N—R′, in which the N (nitrogen)atom is connected to the spacer, and where R and R′ may be,independently: H; a linear, branched, or cyclic alkyl group containingfrom 1 to 10 C atoms and facultatively incorporating with one or moreheteroatoms such as N, O, S, Si, and halogen, as well as multiple bondsamong pairs of atoms; or part of ring structure consisting of 3 to 10carbon atoms and facultatively incorporating one of more heteroatomssuch as N, O, S, Si, or halogen, as well as multiple bonds among pairsof atoms.

Examples of such R and R′ include, but are not limited to, thefollowing:

H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl,benzyl,

(b) a protonatable, nitrogen-containing heterocyclic system such as apyrrolidine, piperidine, pyridazine, morpholine, thiomorpholine,quinuclidine, imidazole, pyridine, and the like, or a substitutedvariant thereof, as illustrated by the following representative, but notexclusive, examples, wherein the wavy line represents the bondconnecting the heterocyclic structure to the spacer:

Representative aspects of formula IIA above thus include, but are notlimited to, the following:

Derivatization of docetaxel with up to three units of Formula IIA, whichmay be different or identical, produces mono-, bis-, or triesters of thetype A, B, C, D, E, F, and G shown below.

Monoester Derivatives of Docetaxel

Diester Derivatives of Docetaxel

Triester Derivative of Docetaxel

In a like mariner, paclitaxel may be converted to mono- and diesterderivatives of the type H, I and J shown below:

Monoester Derivatives of Paclitaxel

Diester Derivative of Paclitaxel

Elaboration of docetaxel or of paclitaxel to such derivatives involves,for instance, the coupling of the unprotected or partially protectedparent drug with a carboxylic acid form of 5 by the use of standardtechniques of modern organic chemistry that are well known to the personskilled in the art.

In some aspects, Z comprises a group of Formula IIB, wherein:

(i) the wavy line represents the bond connecting Formula IIB to theappropriate reactive group, such as an O atom, in the drug [D] (e.g.,compounds 3 and/or 4);

(ii) the “spacer” [S] is as detailed above for Formula IIA

(iii) the solubilizing unit [N] is as detailed above for Formula IIA

(iv) R′ is H, or a linear, branched, or cyclic alkyl group containingfrom 1 to 10 C atoms and facultatively incorporating with one or moreheteroatoms such as N, O, S, Si, and halogen, as well as multiple bondsamong pairs of atoms.

Examples of such R′ include, but are not limited to, the following:

H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl,benzyl,

Representative aspects of structure Formula IIB above include, but arenot limited to, the following:

Derivatization of docetaxel with up to three units of the type 6, whichmay be different or identical, produces mono-, bis-, or tricarbamates ofthe type A, B, C, D, E, F, and G shown below.

Monocarbamate Derivatives of Docetaxel

Dicarbamate Derivatives of Docetaxel

Tricarbamate Derivative of Docetaxel

In a like manner, paclitaxel may be converted to mono- and dicarbamatederivatives of the type H, I and J shown below:

Monocarbamate Derivatives of Paclitaxel

Dicarbamate Derivative of Paclitaxel

Elaboration of docetaxel or of paclitaxel to such derivatives involves,for instance, the coupling of the unprotected or partially protectedparent drug with an isocyanate (R″═H) or an imidazolide (R″≠H) form of 6by the use of standard techniques of modern organic chemistry that arewell known to the person skilled in the art.

In some aspects, Z is a group of the general Formula IIC, wherein:

(i) the wavy line represents the bond connecting Formula IIC to anappropriate reactive group, such as an O atom, in the drug [D] (e.g.,compounds 3 and/or 4);

(ii) the “spacer” is as detailed above for Formula IIA

(iii) Group [N] is as detailed above for Formula IIA

(iii) R^(A) and R^(B) represent, independently, a linear, branched, orcyclic alkyl group containing from 1 to 10 C atoms and facultativelyincorporating with one or more heteroatoms such as N, O, S, Si, andhalogen, as well as multiple bonds among pairs of atoms.

Examples of such R^(A) and R^(B) include, but are not limited to, thefollowing: H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,phenyl, and benzyl.

Representative aspects of Formula IIC above include, but are not limitedto, the following:

Derivatization of docetaxel with up to three units of Formula IIC, whichmay be different or identical, converts it to mono-, bis-, or tris-silylethers of the type A, B, C, D, E, F, and G shown below.

Mono-Silyl Ether Derivatives of Docetaxel

Bis-Silyl Ether Derivatives of Docetaxel

Tris-Silyl Ether Derivatives of Docetaxel

In a like manner, paclitaxel may be converted to mono- and diesterderivatives of the type H, I and J shown below:

Mono-Silyl Ether Derivatives of Paclitaxel

Bis-Silyl Ether Derivative of Paclitaxel

Elaboration of docetaxel or paclitaxel to such derivatives involves, forinstance, the coupling of the unprotected or partially protected parentdrug with a chloride or an imidazolide form of Formula IIC by the use ofstandard techniques of modern organic chemistry that are well known tothe person skilled in the art.

The technology exemplified above with taxanes is applicable to any drugpossessing suitable anchoring sites, such as OH, COOH (carboxyl), or NHgroups, for solubilizing units, [Z], of Formulae IIA, IIB, or FormulaeIIC.

In some aspects, the drug is etoposide, which is a widely usedanticancer agent approved for the treatment of lymphoma, lung andtesticular cancers. Etoposide exhibits poor water-solubility, undergoesmetabolic inactivation, and has substantial toxic side effects. Invarious preferred aspects, etoposide LN formulations provided hereinhave substantially reduced toxicity, improved solubility andbioavailability, and increased efficacy.

To illustrate, etoposide, 8, and the corticosteroid prednisone, 9, maybe converted to ester, carbamate, or silyl ether derivatives as detailedabove for the taxanes. [Z] in these derivatives is as defined earlierfor compounds in the taxol series

In a like manner, cyclosporin, 10, azathioprine, 11, etc., may beconverted to derivatives that are suitable for liposomal formulation:

In some aspects, a drug of interest is derivatized with alipid-solubilizing unit that comprises a weakly basic amine group and alipophilic group. In some preferred aspects, the solubilizing unit has astructure that similar to that of the lipids comprising the liposomalmembrane. For example, in some aspects a drug derivative is of thegeneral formula: [D]-[L]-[S]-[N], wherein [S] is a Spacer as definedabove in relation to Formulae IIA-IIC and [N] is a solubilizing domainand [L] is a linker, as defined below.

In some aspects, [N] is a group of Formula IVA (“internal” derivatives)or Formula IVB (“terminal” derivatives), wherein:

(i) A represents a carbonyl group (C═O); a carbamoyl group (NR—C═O,where R is H or an alkyl group incorporating from 1 to 5 C atoms); or amethylene group (CH₂);

(ii) R¹ and R² represent a linear or branched lipophilic alkyl groupcontaining up to 30 carbon atoms and facultatively incorporating one ormore multiple bonds between pairs of adjacent carbon atoms;

(iii) R³ and R⁴ represent, independently, H; or alkyl groupsincorporating from 1 to 5 C atoms, such as methyl ethyl, propyl,isopropyl, butyl, isobutyl, etc.; or branches of a ring structure suchas pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, etc.

(iii) the Linker, [L], is:

(a) a carbonyl group, C═O;

(b) a carbamoyl group, NR—C═O, where R is H or an alkyl groupincorporating from 1 to 5 C atoms; OR

(c) a group R^(A)—Si—R^(B) as defined above.

Below are a number of clinically significant taxanes derivatized with asolubilizing group comprising a weakly basic moiety and a lipophilicmoiety, to form “terminal” type (Formula IVB) derivatives and “internal”type (Formula IVA) derivatives. Such derivatives can be made by, forinstance, coupling any unprotected or partially protected parent drugwith a carboxylic acid, an acyl imidazolide, a carbamoyl imidazolide, asilyl chloride, or a silyl imidazolide form of the linker, [L], usingtechniques known in the art. The resulting derivatives can be activelyloaded into LN such that the drug resides in the liposomal membrane.

Docetaxel derivatives of Formula IVA include esters, carbamates, andsilyl ethers with the following representative structures:

Docetaxel Monoesters, Monocarbamates, and Monosilyl Ethers of FormulaIVA

Paclitaxel derivatives of Formula IVA include esters, carbamates, andsilyl ethers with the following representative structures:

Paclitaxel Monoesters, Monocarbamates, and Monosilyl Ethers of FormulaIVA

Docetaxel derivatives of Formula IVB include esters, carbamates, andsilyl ethers of the general type described earlier, but possessing thefollowing representative structures:

Docetaxel Monoesters, Monocarbamates, and Monosilyl Ethers of FormulaIVB

Paclitaxel derivatives of Formula IVB include esters, carbamates, andsilyl ethers of the general type described earlier, but possessing thefollowing representative structures:

Paclitaxel Monoesters, Monocarbamates, and Monosilyl Ethers of FormulaIVB

Similarly, etoposide, 8, prednisone, 9, cyclosporin, 10, azathioprine,11, and other drugs may be derivatized with a solubilizing unit thatcomprises a weakly basic group and a lipophilic group, such that thedrug can be actively loaded within the LN membrane. With regard tocompounds 8-11, 13 and 14 refer to derivatives of the formula[D]-[L]-[S]-[N] and 15 and 16 are derivatives of the formula[L]-[S]-[N], wherein [N] is according to Formula IVB in type 13 andFormula IVA in type 14 and [L] and [S] are as described above.

In some aspects, the drug is a new chemical entity (NCE) selected, e.g.,from a combinatorial library, for therapeutic efficacy and one or moreproperties, such as lipophilicity and/or low aqueous solubility, thatwould interfere with the pharmaceutical utility of the drug absent theinstant methods.

In further aspects, drug derivatives are prepared by modifying a newlydiscovered and/or characterized drug (e.g., a new chemical entity(NCE)). Often pharmacologically potent hits from chemical libraryscreens prove to be less than ideal candidates for pharmaceuticaldevelopment and use. For example, solubility issues are the main reasonsthat most NCEs do not advance in development and are discarded. Thechemistry platform outlined herein enables development and use of suchcompounds by specifically modifying them with weak-base chemicalmoieties that promote formulation in LN using known methods. Theintegration of high-throughput combinatorial chemistry methods forgenerating and screening drug candidates with the medicinal chemistryplatform described herein provides an alternative approach for thedevelopment of drugs (and diagnostic agents) that can replace existingdrug development strategies predicated on finding compounds withdrug-like properties.

Accordingly, in some aspects, methods are provided herein foridentifying drug candidates having a therapeutic activity of interestand low aqueous solubility, lipophilicity, and/or other properties thatwould prevent or interfere with use of the free compound and/or preventefficient loading of the compound into LN. For example, in some aspects,methods are provided comprising the steps of: screening a population ofcompounds produced through combinatorial chemistry to identify drugcandidates having a therapeutic activity of interest, and screening thedrug candidates for one or more additional properties to identifycandidates for derivatization according to methods described herein. Infurther aspects, the candidates for derivatization are derivatized witha weakly basic group, actively loaded into LN, and the LN are screenedto identify formulation candidates having a desired therapeuticactivity. Advantageously, screening methods provided herein identifydrug candidates for use in LN formulations that would otherwise not bedetected using standard methods.

Liposomes used in methods and compositions provided herein can be formedfrom standard vesicle-forming lipids, which generally include neutraland negatively or positively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of, e.g., liposome size, stability of the liposomes in thebloodstream, the desired release rate, and other factors known in theart.

In some aspects, the major lipid component of liposomes used in methodsand compositions described herein is phosphatidylcholine.Phosphatidylcholines having a variety of acyl chain groups of varyingchain length and degree of saturation may be used. In some aspects,phosphatidylcholines containing saturated fatty acids with carbon chainlengths in the range of C₁₄ to C₂₂ are preferred. Saturated long-chainphosphatidylcholines are less permeable and more stable in vivo thantheir unsaturated counterparts. Phosphatidylcholines with mono- ordi-unsaturated fatty acids and mixtures of saturated and unsaturatedfatty acids may also be used. Other suitable lipids include, e.g.,etherlipids in which the fatty acids are linked to glycerol via etherlinkages rather than ester linkages. Liposomes used herein may also becomposed of sphingomyelin or phospholipids with head groups other thancholine, such as ethanolamine, serine, glycerol, phosphatidic acid andinositol.

In some preferred aspects, liposomes include a sterol, preferablycholesterol, at molar ratios of from 0.1 to 1.0(cholesterol:phospholipid). Examples of preferred liposome compositionsinclude distearoylphosphatidylcholine/cholesterol,dipalmitoylphosphatidylcholine/cholesterol,dimyrystoylphosphatidylcholine/cholesterol and eggsphingomyelin/cholesterol.

In other aspects, liposomes can contain negatively or positively chargedlipids. Examples of useful negatively charged lipids include, but arenot limited to dimyrystoyl, -dipalmitoyl- anddistearoylphasphatidylglycerol, dimyrystoyl, -dipalmitoyl- anddipalmitoylphosphatidic acid, dimyrystoyl, -dipalmitoyl- anddipalmitoylphosphatidylethanolamine, their unsaturated diacyl and mixedacyl chain counterparts as well as cardiolipin. Not limiting examples ofpositively charged lipids include N,N′-dimethyl-N,N′-dioctacyl ammoniumbromide (DDAB) and chloride DDAC),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),3β-[N—(N′,N′-dimethylaminoethyl)carbamoyl)cholesterol (DC-chol),1,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP),1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium chloride (DORI)and cationic lipids described in e.g. B. Martin, M. Sainlos, A.Aissaoui, N. Oudrhiri, M. Hauchecorne, J.-P. Vigneron, J.-M. Lehn and P.Lehn The design of cationic lipids for gene delivery. CurrentPharmaceutical Design 2005, 11, 375-394.

In further aspects, liposomes used herein are coated with a polymerlayer to enhance stability of the LN in vivo (e.g., stericallystabilized liposomes). For example, in some embodiments, LN are formedfrom liposomes containing polyethylene glycol)-conjugated lipids(PEG-lipids) that form a hydrophilic surface layer that improves thecirculation half-life of LN and enhances the amount of LN that reachtherapeutic targets, such as a site of infection or a tumor site. Thegeneral approach is described, e.g., in Working et al. J Pharmacol ExpTher, 289: 1128-1133 (1999); Gabizon et al., J Controlled Release 53:275-279 (1998); AdlakhaHutcheon et al., Nat Biotechnol 17: 775-779(1999); and Koning et al., Biochim Biophys Acta 1420: 153-167 (1999).Examples of useful PEG-lipids include, but are not limited to,1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-350] (mPEG 350 PE);1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-550] (mPEG 550 PE);1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-750] (mPEG 750 PE);1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-1000] (mPEG 1000 PE);1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000] (mPEG 2000 PE);1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-3000] (mPEG 3000 PE);1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-5000] (mPEG 5000 PE); N-Acyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol) 750] (mPEG 750 Ceramide);N-Acyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol) 2000] (mPEG2000 Ceramide); and N-Acyl-Sphingosine-1-[Succinyl(Methoxy PolyethyleneGlycol) 5000] (mPEG 5000 Ceramide).

A variety of methods are available for preparing liposomes as described,e.g., in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); U.S.Pat. Nos. 4,235,871, 4,501,728, and 4,837,028; Liposomes, Marc J. Ostro,ed., Marcel Dekker, Inc., New York, 1983, Chapter 1; and Hope, et al.,Chem. Phys. Lip. 40:89 (1986), all of which are incorporated herein byreference. In some preferred aspects, the liposomes are small,approximately 100 nm in diameter liposomes generated by extrudinghydrated lipid dispersions through filters with 100 nm pores, asdescribed generally in Hope et al., Biochim. Biophys. Acta, 812: 55-65(1985), incorporated herein by reference.

In one method, multilamellar vesicles of heterogeneous sizes areproduced by dissolving vesicle-forming lipids in a suitable organicsolvent or solvent system and drying the mixture under vacuum or aninert gas to form a thin lipid film. Alternatively, the lipids may bedissolved in a suitable solvent, such as tertiary butanol, and thenlyophilized to form a more homogeneous lipid mixture. The film or powderis covered with an aqueous buffered solution of a monovalent or divalentmetal ion and allowed to hydrate, typically over a 15-60 minute periodwith agitation. The size distribution of the resulting multilamellarvesicles can be shifted toward smaller sizes by hydrating the lipidsunder more vigorous agitation conditions or by adding solubilizingdetergents such as deoxycholate. In another method, the lipids aredissolved in a water-miscible organic solvent such as ethanol and thencombined with the aqueous buffer to form a multilamellar liposomesuspension. Alternatively, the lipids are dissolved in awater-immiscible organic solvent, mixed with the aqueous medium andliposomes formed by evaporation of the organic solvent.

Several techniques are available for sizing liposomes to a desired size.One sizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles less than about 0.05 microns in size.Homogenization or microfluidization are other methods which rely onshearing energy to fragment large liposomes into smaller ones. In atypical homogenization procedure, multilamellar vesicles arerecirculated through a standard emulsion homogenizer until selectedliposome sizes, typically between about 0.1 and 0.5 microns, areobserved. In both methods, the particle size distribution can bemonitored by conventional laser-beam particle size discrimination.

Extrusion of liposomes through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is a very effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size.

In some aspects, methods are provided for loading a weak base derivativeinto liposomes using an active loading technique. In some aspects,liposomes are loaded by imposing a pH gradient across the liposomemembrane (wherein the liposome interior is acidic) and incubating theliposome with the drug to be encapsulated, as described, e.g., inMaurer, N., Fenske, D., and Cullis, P. R. (2001) Developments inliposomal drug delivery systems. Expert Opinion in Biological Therapy 1,923-47; N. L. Boman, D. Masin, L. D. Mayer, P. R. Cullis and M. B. Bally(1994) “Liposomal Vincristine Which Exhibits Increased Drug Retentionand Increased Circulation Longevity Cures Mice Bearing P388 Tumors”,Cancer Res. 54, 2830-2833; D. N. Waterhouse, T. D. Madden, P. R. Cullis,M. B. Bally, L. D. Mayer, M. Webb, Preparation, characterization, andbiological analysis of liposomal formulations of vincristine. MethodsEnzymol. 391 (2005) 40-57, hereby incorporated by reference. In somepreferred aspects, the pH gradient is an ammonium sulfate gradient, asdescribed generally in G. Haran, R. Cohen, L. K. Bar, Y. Barenholz,Transmembrane ammonium sulfate gradients in liposomes produce efficientand stable entrapment of amphipathic weak bases. Biochim. Biophys. Acta1115 (1993) 201-215 and U.S. Pat. No. 5,316,771, hereby incorporated byreference. Once the drug has been loaded into the liposomes, thecompositions can be used directly, or the composition can be furthertreated to remove any unloaded drug.

pH loading techniques generally involve two steps, the generation of thepH gradient with low intraliposomal pH and the subsequent loading of thedrug. Transmembrane proton gradients can be generated by a variety ofways. Liposomes can be prepared in a low pH buffer such as a pH 4citrate buffer followed by exchange of the external buffer solutionagainst a pH 7.5 buffer (e.g. Madden et al., Chem. Phys. Lipids,53:37-46 (1990)). Alternatively, ionophores can be used in conjunctionwith cation gradients (high internal cation concentrations) (e.g. Fenskeet al., Biochim Biophy. Acta, 1414:188-204 (1998)). Ionophores such asnigericin and A23187 couple the outward movement of monovalent ordivalent cations, respectively, to the inward movement of protons thusacidifying the liposome interior. Furthermore, liposomes can be preparedin the presence of high concentrations of a weak base such as ammoniumsulfate (Haran et al., Biochim. Biophys. Acta, 1151:201-215 (1993)).Removal of the external ammonium salt solution results in the generationof a pH gradient according to the same principle, which is alsoresponsible for the subsequent drug loading process. The ammoniumsulfate loading technique does not require a large pH gradient toachieve efficient loading, as the loading process is sustained by anexchange of the two different amines (drug goes in, ammonia comes out)and hence works well at very low external pH. This is an advantage if,for example, the drug is unstable or insoluble at neutral pH. Inaddition to pH gradients, metal ion gradients can be used for activeloading (e.g. Cheung et al., Biochim Biophys Acta, 1414:205-216 (1998)).This loading method relies the same basic principles as the pH gradienttechnique. The neutral form of the weak base drug can permeate acrossthe membrane and is retained in the aqueous interior of the liposomesthrough formation of a drug-metal ion complex.

For loading of water-soluble weak base drugs into LN, the drug can bedissolved in an aqueous solution (e.g. 300 mM sucrose, or isotonicbuffer solutions with appropriate pH), combined with the liposomesuspension and then incubated at appropriate temperature. The drugsolution can contain a small (non-membrane permeabilizing) amount of awater-miscible organic solvent to increase the solubility of the drug(e.g. <10% ethanol). The incubation temperature and time depend on thelipid composition and the nature of the drug. Typically, liposomescomposed of cholesterol and long-chain saturated fatty acids such asDSPC/chol LN are less permeable than LN formed from short-chainsaturated lipids (e.g. DMPC/chol) or unsaturated lipids and requirehigher temperatures to achieve rapid and efficient loading. For example,DSPC/chol LN typically require temperatures equal or higher than 60° C.;loading is typically complete after 5-15 minutes, but may take up to 2hours.

For loading of lipophilic weak base drugs, the drug can be treated likea lipid. For example, lipids and drug can be co-mixed and liposomesformed as described above; the lipophilic drug is then distributedbetween the two monolayers of the liposome bilayer. The drug in theexternal monolayer is then loaded into the liposome interior (flipped tothe inner monolayer of the LN bilayer) in response to a trans-membranepH or other ion gradient using the methods described above.

In additional aspects, pharmaceutical compositions are providedcomprising a LN formulation provided herein. Also provided herein aremethods for treating a disease or condition, comprising administering aLN composition provided herein. In yet further aspects, kits areprovided comprising an LN composition described herein and instructionalmaterial teaching the methodologies and uses of the invention, asdescribed herein.

Pharmaceutical compositions comprising the liposomes and compounds ofthe invention are prepared according to standard techniques, as well asthose techniques described above. Preferably, the pharmaceuticalcompositions are administered parenterally, i.e., intraanicularly,intravenously, subcutaneously, or intramuscularly. More preferably, thepharmaceutical compositions are administered intravenously by a bolusinjection or infusion. Suitable formulations for use in the presentinvention are found in Remington's Pharmaceutical Sciences, MackPublishing Company, Philadelphia, Pa., 17th ed. (1985).

Preferably, the pharmaceutical compositions are administeredintravenously Thus, this invention provides compositions for intravenousadministration which comprise liposomes suspended in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g., water, buffered water, 0.9% isotonic saline, and thelike. These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous suspension may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous solutionprior to administration. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, sodium phosphate, polyethylene glycol(PEG₄₀₀), etc.

The concentration of liposomes, in the pharmaceutical formulations canvary widely, i.e., from less than about 0.5 mg/mL lipid, usually at orat least about 10-50 mg/mL lipid to as much as 100 mg/mL lipid or higherand will be selected primarily by fluid volumes, viscosities, stability,drug dose required, etc., in accordance with the particular mode ofadministration selected.

Liposome charge is an important determinant in liposome clearance fromthe blood, with negatively charged liposomes being taken up more rapidlyby the reticuloendothelial system (Juliano, Biochem. Biophys. Res.Commun. 63:65 1 (1975)) and thus having shorter half-lives in thebloodstream. Liposomes with prolonged circulation half-lives aretypically desirable for therapeutic and diagnostic uses, where liposomeshave to accumulate at distal disease sites such as tumors. For instance,liposomes which have circulation half-lives from 2, 8, 12, or up to 24hours are particularly preferred.

Additionally, the liposome suspension may include lipid-protectiveagents which protect lipids against free-radical and lipid-peroxidativedamages on storage. Lipophilic free-radical quenchers, such asalpha-tocopherol and water-soluble iron-specific chelators, such asferrioxamine, or an anti-oxidant such as ascorbic acid are suitable.

The following examples are provided by way of illustration and arenon-limiting.

EXAMPLES Example 1 Chemical Synthesis Methods

Weak base derivatives and unmodified drugs were quantitated by ultrahigh performance liquid chromatography (UPLC). The instrument consistedof a Waters® Acquity™ UPLC system equipped with a photodiode arraydetector (PDA) and a triple-quad (TQ) MS detector; Empower™ dataacquisition software version 2.0 was used (Waters, USA). Separationswere performed using a Waters® Acquity™ BEH C18 column (1.7 μm, 2.1×100mm) at a flow rate of 0.25 mL/min, with mobile phases A and B consistingof water with 0.1% trifluoroacetic acid (TFA) and acetonitrile with 0.1%TFA, respectively. For prednisone and etoposide derivatives andunmodified drugs the mobile phases consisted of water with 0.1% formicacid (A) and acetonitrile with 0.1% formic acid (B). The mobile phaseswere delivered at a programmed linear gradient at a column temperatureof 23° C.

For docetaxel derivatives and docetaxel, separation was initiated with amobile phase ratio of 50:50 (A:B). The ratio was changed to 10:90 (A:B)over a period of 2 min using a linear curve and then maintained at 10:90(A:B) over a period of 0.5 min. The mobile phase was subsequentlychanged back to 50:50 (A:B) over a period of 0.1 min and this ratio wasmaintained for 0.4 min before the next sample was injected. Forprednisone derivatives and prednisone, separation was initiated with amobile phase ratio of 80:20 (A:B). The ratio was changed to 40:60 (A:B)over a period of 4 min using a linear curve and then to 10:90 (A:B) overa period of 0.1 min. The latter ratio was maintained for 0.4 min. Themobile phase was subsequently changed back to 80:20 (A:B) during a spanof 0.1 min and this ratio was maintained for 0.9 min before the nextsample was injected. For the etoposide derivative and etoposide,separation was initiated with a mobile phase ratio of 80:20 (A:B). Theratio was changed to 72.5:27.5 (A:B) over a period of 1 min using alinear curve, then to 60:40 (A:B) over a period of 3 min and 10:90 (A:B)over a 0.1 min period. This ratio was maintained for 0.4 min. The mobilephase was subsequently changed back to 80:20 (A:B) during a span of 0.1min and this ratio was maintained for 0.4 min before the next sample wasinjected.

The analyte was detected by a PDA and TQ-MS detector at a wavelength of230 nm (in the case of docetaxel and docetaxel derivatives) and 254 nm(for prednisone and etoposide derivatives) and ES⁺ ion mode with a conevoltage of 30V, respectively. LN formulated derivatives were solubilizedin TFA- or formic acid-acidified ethanol (0.1% vol.). For detection ofLN-formulated drugs within blood plasma samples, 50 μL plasma was addedto 150 μL methanol acidified with TFA or formic acid (0.1% v/v) and themixture was centrifuged at 4° C. for 30 min at 10,000×g to pellet theprecipitated proteins. Acidification of methanol was necessary tostabilize the prodrugs. The limit of MS detection (LOD) for docetaxeland docetaxel derivative (TD-1) was between about 1-50 ng/mL whenTFA-acidified methanol was used. The limit can be decreased to sub nMconcentrations if needed by using formic acid in place of TFA.

Unless otherwise indicated, ¹H and ¹³CNMR spectra were recorded at roomtemperature on Bruker models AV-300 (300 MHz for ¹H and 75 MHz for ¹³C)and AV-400 (400 MHz for ¹H and 100 MHz for ¹³C). Chemical shifts arereported in parts per million (ppm) on the 8 scale and couplingconstants, J, are in hertz (Hz). Multiplicities are described as “s”(singlet), “d” (doublet), “t” (triplet), “q” (quartet), “dd” (doublet ofdoublets), “dt” (doublet of triplets), “m” (multiplet), “b” (broad).Low-resolution mass spectra (m/z) were obtained in the electrospray(ESI) mode.

LN formulated derivatives were viewed by Cryo-TEM performed with aTecnai G2 20 TWIN Mk. 2 Transmission Electron Microscope (CDRD Imaging,Vancouver, Canada). The Instrument was operating at 200 kV inbright-field mode. Digital images were recorded under low doseconditions with a FEI Eagle 4k HR CCD camera and analysis software FEITIA. An underfocus of 1-3 μm was used to enhance image contrast. Samplepreparation was done with a Vitrobot Mark IV vitrification robot onLacey Formvar 300 grids (#01890 from Ted Pella).

All reagents and solvents were commercial products and were used withoutfurther purification. Flash chromatography was performed on Silicycle230-400 mesh silica gel. Analytic and preparative TLC was carried outwith Merck silica gel 60 plates with fluorescent indicator. Spots werevisualized with UV light, KMnO₄ or p-anisaldehyde.

General Synthetic Strategy

A general strategy provided herein (FIG. 10) involves the derivatizationof a water-insoluble drug 1 that contains an appropriate anchoring site,such as an OH or an NH group, with a properly tailored solubilizing unitrepresented by the general structure 2. The general scheme also appliesto the synthesis of lipophilic weak base drug derivatives. The resultantwater-soluble conjugate 3 can be loaded into LN using a pH or iongradient as the driving force. The derivative 3 is either active byitself and/or is rapidly converted into the active parent drug 1 underphysiological conditions.

The technology is based on a number of physical properties of 3, such as(i) water solubility; (ii) pKa of the protonated nitrogen functionality;(iii) stability under liposome loading conditions; (iv) rate of releaseof the free drug under physiological conditions. In turn, theseproperties are a function of the nature of the linker, of the spacer,and of groups R¹ and R² in 2.

In some aspects, the solubilizing units comprise a carboxy linker group,a spacer such as n-C₁-C₄ chain, and an amine group such asN-methylpiperazine, morpholine, piperidine, pyrrolidine, ordimethylamine. Exemplary solubilizing units include:

where n is between 1 and about 10, or more preferably 1 and 4.

Example 2 Taxane Derivatives

Docetaxel was derivatized at the hydroxyl group in the C-2′ positionwith N-methyl-piperazinyl butanoic acid to form an amino ester prodrug(TD1), as described below.

2′-O-(N-methyl-piperazinyl butanoic acid ester) derivative of docetaxel(TD 1)

Linker Synthesis: 4-(4-methylpiperazin-1-yl)butanoic acid hydrochloride

1-methyl piperazine (7.68 mL, 70 mmol, 4 equivalents) was added to astirred solution of ethyl 4-bromobutanonate (2.5 mL, 17.3 mmol) in ethylacetate (50 mL) at room temperature. The solution was stirred at 25° C.for 1 h with evolution of a white precipitate, and then heated on an oilbath to 70° C. for 1 h. TLC analysis (20% ethylacetate (EtOAc) inhexanes, Rf=0.9 (starting material), 0.1 (product), visualized withiodine, 12) indicated complete consumption of the bromide reagent. Thereaction was diluted with EtOAc (100 mL), transferred to a separatoryfunnel and the organic phase washed with water (100 mL), sodiumbicarbonate (NaHCO₃, saturated, 2×100 mL), brine (100 mL), dried overmagnesium sulfate (MgSO₄) and concentrated to give a slightly yellowoil. The oil was dissolved in methylene chloride (20 mL) and loaded ontoa pre-equilibrated plug of silica gel (20% EtOAc in hexanes, 150 mLSiO₂). The desired product was eluted from the silica with increasinglypolar eluent (first with 20% EtOAc in hexanes, then with 5-25% MeOH(containing 5% NH₄OH) in EtOAc).

Fractions containing the desired material were pooled and concentratedto give ethyl-4-(4-methylpiperazin-1-yl)butanoate (3.63 g,quantitative). Water (20 mL) and hydrochloric acid (HCl, 10M, 20 mL, 10equivalents) were added to the flask containing the resulting oil. Theflask was fitted with a reflux condenser and heated at 110° C. for 3 h.The reaction mixture was then allowed to cool to room temperature andwas subsequently concentrated under vacuum until there was only an oilyresidue remaining. The residue was re-dissolved in distilled water andthe concentration process repeated. The remaining syrup was dissolved inethanol (50 mL) at 85° C. Addition of a small quantity of water (˜1 mL)was required to dissolve all solids (adding larger volumes of water willadversely affect yield). The solution was allowed to stand at roomtemperature for 3 h and was then transferred to a refrigerator (5° C.)for 16 h. The precipitate was filtered off, transferred to a pre-weighedvial and placed in a desiccator over Drierite at high vacuum for 16 h togive 4-(4-methylpiperazin-1-yl)butanoic acid hydrochloride as acrystalline and non hygroscopic material (3.02 g, 80% based on the monoHCl salt).

1H NMR (D₂O, 400 MHz) δ (ppm)=3.60 (br s, 8H), 3.26-3.22 (m, 2H), 2.93(s, 3H), 2.43 (t, J=7.0 Hz, 2H), 1.99-1.91 (m, 2H). ¹³C NMR (D2O, 100MHz) δ (ppm)=176.5, 55.9, 50.2, 48.7, 42.8, 30.3, 18.7.

Esterification and Salt Formation: TD-1 Hydrochloride Salt

Triethylamine (NEt₃, 10.0 mL, 5 equivalents) was added to a stirredsolution of docetaxel (3.997 g, 4.95 mmol) and4-(4-methylpiperazin-1-yl)butanoic acid hydrochloride (1.213 g, 5.44mmol, 1.1 equivalents) in dichloromethane (CH₂Cl₂, 60 mL). The reactionvessel was then cooled in an ice bath and Mukaiyama reagent(2-chloro-1-methylpyridinium iodide, 1.667 g, 6.53 mmol, 1.32 equiv) wasadded. The solution went yellow with the dissolution of the pyridiniumsalt. The flask was removed from the ice bath after 30 minutes and thereaction was allowed to proceed for an additional 16 h. TLC indicatedgood, but incomplete conversion of the starting material to the desiredproduct (8% MeOH (with 5% NH₄OH) in CH₂Cl₂, stained with 5% H₂SO₄ inethanol). An additional 0.5 equivalents of the pyridinium salt (0.632 g,0.5 equiv) and amino acid (0.120 g, 0.1 equiv) was added to theice-cooled solution while stirring. After 3 h the reaction mixture wasconcentrated on a rotary evaporator at high vacuum to yield a slightlyorange solid. The solid was dissolved in CH₂Cl₂ (150 mL) and EtOAc (20mL), transferred to a separatory funnel and partitioned between theorganic phase and a saturated NaHCO₃ solution (100 ml). The organicphase was then washed with brine (100 mL), dried over MgSO₄, filteredand concentrated to give a slightly golden syrup. The syrup wasdissolved in CH₂Cl₂ (20 mL), loaded onto a pre-equilibrated silica gelcolumn (4% MeOH (with 5% NH₄OH) in CH₂Cl₂, 250 mL, 40 mm diameter) andeluted with increasingly polar solvent (4-10% MeOH (with 5% NH₄OH) inCH₂Cl₂, 2% increments, 500 mL/increment).

The fractions containing the desired material were collected andconcentrated to yield 3.8909 g (80.5%) of compound. ¹H NMR analysis ofthe compound indicated good purity, with the presence (˜10%) of peaksattributed to a regioisomer. The material was re-dissolved in CH₂Cl₂ andsubjected to the same chromatographic conditions described above, using1% increments of MeOH in CH₂Cl₂ from 5-10% (500 mL/increment). Fractionscontaining pure material were identified by TLC, collected andconcentrated to give 2.96 g of compound with a clean NMR spectrum.

The material was dissolved in 2-propanol (45 mL) and HCl (6.3 mL, 1M indiethylether (Et₂O), 2.05 equivalents) was added dropwise under cooling(0° C.) to generate the hydrochloride salt. The suspension wasconcentrated to dryness, and the resulting cream colored solid was driedat high vacuum and re-crystallized from 2-propanol (45 mL) by theaddition of Et₂O (10 mL). The precipitate was filtered off on a Buchnerfunnel and dried at high vacuum for 18 h yielding ˜2.5 g. The docetaxelderivative (TD-1) was characterized by NMR, mass spectroscopy, elementalanalysis and UHPLC-UV to confirm identity and purity. Chromatographicpurity by UHPLC-UV was 96.7%.

¹H NMR (400 MHz, D₂O): δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.65 (t, J=7.4Hz, 1H), 7.56 (t, J=7.7 Hz, 2H), 7.43-7.37 (m, 4H), 7.25 (br t, J=6.2Hz, 1H), 6.09 (m, 1H), 5.61 (d, J=7.1 Hz, 1H), 5.32 (m, 1H), 5.27-5.24(m, 2H), 4.99 (d, J=8.1 Hz, 1H), 4.22-4.13 (m, 3H), 3.83 (br d, J=6.8Hz, 1H), 3.61 (s, 1H), 3.21-3.09 (m, 2H), 2.67-2.33 (m, 6H), 2.23-2.17(m, 1H), 2.12-1.98 (m, 2H), 1.97-1.76 (m, 5H), 1.68 (s, 3H), 1.40 (s,9H), 1.15 (s, 3H), 1.11 (s, 3H).

¹³C NMR (100 MHz, D₂O): δ (ppm)=211.22, 173.26, 172.48, 170.57, 167.58,157.22, 138.72, 136.13, 134.48, 129.96, 129.29, 129.00, 128.77, 126.95,84.49, 80.87, 78.51, 76.55, 75.55, 75.04, 74.26, 72.65, 71.28, 57.39,55.59, 50.32, 48.78, 46.29, 42.83, 42.68, 35.25, 34.73, 30.13, 29.83,29.60, 27.51, 25.900, 23.69, 22.43, 20.75, 18.77, 16.78, 13.60, 9.55.

Elemental analysis: calculated based on TD-1+2HCl+1H₂O: C, 58.53; H,6.90; Cl, 6.65; N, 3.94; Obs.: C, 58.50; H, 6.97; Cl, 6.58; N, 4.13;HPLC/MS (m/z); 977.4 (m+H), 96.7% area by UPLC-UV.

TD1 Analogs

TD1 contains a dibasic amino acid ester at O-2′ that is believed toassist in the directed release of the parent compound (docetaxel)through neighboring group participation. A series of TD 1 analogs weresynthesized as described below. The analogues have variations in thechain length of the amino-acyl linker and the structure of the basicamino-acyl moiety designed to modulate the rate of ester hydrolysis viaanchimeric assistance (Pop et al., Pharmaceutical Research,13(3):469-475 (1996); Rautio et al., J. Med. Chem., 43(3):1489-1494(2000)), allowing the rate at which the parent compound is released tobe fine-tuned for various therapeutic applications.

For a ring closing reaction, 3-7 membered ring transition states arefavored when the reaction centre is sp² hybridized, as in the case ofintramolecular ester hydrolysis. There are two possible modes ofhydrolysis: Mode A, in which the amine acts directly at the carbonyl togenerate the parent drug and an activated acyl-ammonium intermediate;and Mode B, in which the amine acts as a general base to increase thenucleophilicity of the solvent (water in this case), thereby increasingthe rate of hydrolysis and ejecting the zwitterionic amino acid. The TD1analogues synthesized below all allow for hydrolysis by Mode A. Onlyshorter amino acid esters (n=1-3) allow hydrolysis by Mode B.

In the first series of analogues, the weak-base solubilization unitcomprises a piperazinyl amino moiety with an alkyl linker of varyinglengths relative to TD1. In the next series of analogues, the same alkyllinkers were used and the amino moiety was varied to include morpholinoand piperidinyl substituents. The amino moieties vary in nucleophilicityaccording to the order: N-methyl piperazine>morpholine>piperidine (e.g.,Baldwin, J. Chem. Soc. Chem. Commun., 734-736 (1976); Baldwin et al., J.Org. Chem., 42(24):3846-3852 (1977)). Basicity is inverted, withN-methyl piperidine having a pK_(a) of 2 units higher than N-methylpiperazine. As such, the N-methyl piperazino compounds are expected tobe more susceptible to Mode A hydrolysis and to require lower pH valuesto achieve protonation.

N-alkylation to amino-ester (general procedure) tert-butyl3-(4-methylpiperazin-1-yl)propanoate

4-methyl piperazine (7.68 mL, 70 mmol, 4 equiv) was added to a stirredsolution of tert-butyl 3-bromopropionate (3.0 mL, 18 mmol) in ethylacetate (15 mL) at 0° C. The solution was stirred at 25° C. for 1 h withevolution of a white precipitate, then heated on an oil bath to 55° C.for 2 h. TLC analysis (20% EtOAc in Hexanes, Rf=0.9 (starting material),0.1 (product)) indicated complete consumption of the bromide reagent.The reaction was diluted with EtOAc (100 mL) and transferred to aseparatory funnel, and the organic phase was washed with water (100 mL),NaHCO₃ (sat'd, 2×100 mL), and brine (100 mL), dried over MgSO₄ andconcentrated to give a slightly yellow oil. The oil was dissolved inmethylene chloride (20 mL), loaded on to a pre-equilibrated plug ofsilica gel (20% EtOAc in Hexanes, 150 mL SiO₂) and the desired productwas eluted from the silica with increasingly polar eluent (EtOAc inhexanes, starting at 20%, 200 mL volumes increasing in 15% increments to100%). Fractions containing the desired material were concentrated togive tert-butyl 3-(4-methylpiperazin-1-yl)propanoate (4.1 g,quantitative). ¹H NMR (400 MHz, CDCl₃) δ (ppm)=3.45 (d, J=3 Hz, 2H),2.64 (t, J=7.3 Hz, 2H), 2.47 (br s, 6H), 2.38 (t, J=7.3 Hz, 2H), 2.25(s, 3H), 1.42 (s, 9H).

The same general procedure was used to prepare the following analogs:

Benzyl 2-(4-methylpiperazin-1-yl)acetate

¹H NMR (400 MHz, CDCl₃) δ (ppm)=7.33-7.27 (m, 5H), 5.15 (s, 2H), 3.25(s, 2H), 2.60 (br s, 4H), 2.48 (br s, 4H), 2.27 (s, 3H).

Ethyl 5-(4-methylpiperazin-1-yl)pentanoate

¹H NMR (400 MHz, CDCl₃) δ (ppm)=4.10 (q, J=7.2 Hz, 2H), 2.43 (br s, 6H),2.35-2.28 (m, 4H), 2.26 (s, 3H), 1.79 (br s, 2H), 1.62 (p, J=7.2 Hz,2H), 1.54-1.46 (m, 2H), 1.23 (t, 7.2 Hz).

Benzyl 2-morpholinoacetate

¹H NMR (400 MHz, CDCl₃) δ (ppm)=7.46-7.30 (m, 5H), 5.16 (s, 2H), 3.74(t, J=4.7 Hz, 4H), 3.25 (s, 2H), 2.58 (t, J=4.7 Hz, 4H).

Tert-butyl 3-morpholinopropanoate

¹H NMR (400 MHz, CDCl₃) δ (ppm)=3.68 (t, J=4.5, $H), 2.63 (t, J=7.3 Hz,2H), 2.44 (t, J=4.5 Hz, 4H), 2.39 (t, J=7.3 Hz, 2H), 1.44 (s, 9H).

Ethyl 4-morpholinobutanoate

¹H NMR (400 MHz, CDCl₃) δ (ppm)=4.11 (q, J=7.1 Hz, 2H), 3.68 (t, j=4.7Hz, 4H), 2.42-2.38 (m, 4H), 2.37-2.31 (m, 4H), 1.80 (p, J=7.3 Hz, 2H),1.24 (q, J=7.1 Hz, 3H).

Ethyl 5-morpholinopentanoate

¹H NMR (400 MHz, CDCl₃) δ (ppm)=4.11 (q, J=7.1 Hz, 2H), 3.68 (t, j=4.7Hz, 4H), 2.42-2.38 (m, 4H), 2.37-2.27 (m, 4H), 1.80-1.73 (m, 2H),1.53-1.45 (m, 2H), 1.24 (q, J=7.1 Hz, 3H).

Hydrolysis to amino acid (general procedure)3-(4-methylpiperazin-1-yl)propanoic acid hydrochloride (TD11)

To a round bottom flask containing tert-butyl3-(4-methylpiperazin-1-yl)propanoate (4.1 g, 18 mmol), was added amagnetic stir bar, water (20 mL) and HCl (10M, 20 mL, 10 equiv). Theflask was fitted with a reflux condenser, placed in an oil bath andheated to a bath temperature of 110° C. for 3 h. No TLC analysis of thisreaction was conducted. The reaction removed from the oil bath andallowed to cool to room temperature. Once the reaction had cooledsufficiently, it was transferred to a rotary evaporator connected to anoil-driven high vacuum pump. The contents of the flask were concentrateduntil pressure was 0.1 mm Hg and there was only an oily residueremaining. The flask was then removed, the contents re-dissolved indistilled water and the evaporation process was repeated, this timeyielding a syrup that foamed when subjected to high vacuum after waterremoval. It should be noted that if the crude material has anysignificant amount of residual HCl, the acid will re-esterify whensubjected to the following conditions for crystallization, adverselyaffecting yield. Ethanol (50 mL) and a magnetic stir bar were added andthe flask was submerged in an oil bath at 85° C. to dissolve the syrup.Even at reflux not all of the material would dissolve so a smallquantity of water (˜1 mL) was added in a dropwise fashion until allsolids dissolved. If excessive amounts of water are added at this timeit will adversely affect yield.

The resulting solution was removed from the oil bath and allowed tostand at room temperature for 3 h before being transferred to arefrigerator (5° C.) for 16 h. The solids were suspended by sonicatingto loosen them from the sides of the flask and filtered on to a filterpaper lined Buchner funnel. The crystals were then transferred to apre-weighed vial which was placed in a desiccator over Drierite, at highvacuum for 16 h to give solid, air-stable, crystalline and nonhygroscopic material (3.07 g, 82% based on the mono HCl salt). ¹H NMR(D₂O, 400 MHz) δ (ppm)=3.6 (br s, 8H), 3.48 (t, J=6.8 Hz, 2H), 2.93 (s,3H), 2.83 (t, J=6.8 Hz, 2H).

The same general procedure was used to prepare the following analogs:

2-(4-methylpiperazin-1-yl)acetic acid hydrochloride (TD2)

¹H NMR (D₂O, 400 MHz) δ (ppm)=3.87 (s, 2H), 3.85-3.35 (br m, 8H), 2.93(s, 3H).

5-(4-methylpiperazin-1-yl)pentanoic acid hydrochloride (TD3)

¹H NMR (D₂O, 400 MHz) δ (ppm)=3.59 (br s, 8H), 3.22 (t, J=7.8 Hz, 2H),2.93 (s, 3H), 2.36 (t, J=7.2 Hz, 2H), 1.75-1.69 (m, 2H), 1.57 (p, J=7.8Hz).

4-morpholinobutanoic acid hydrochloride (TD4)

¹H NMR (D₂O, 400 MHz) δ (ppm)=4.02 (br d, J=12.3 Hz, 2H), 3.73 (br t,J=12.3 Hz, 2H), 3.46 (br d, J=12.3 Hz, 2H), 3.15-3.06 (m, 4H), 2.41 (t,J=7.1 Hz, 2H), 1.97-1.89 (m, 2H).

2-morpholinoacetic acid hydrochloride (TD5)

¹H NMR (D₂O, 400 MHz) δ (ppm)=4.10-3.70 (m, 6H), 3.50 (br s, 2H), 3.2(br s, 2H).

3-morpholinopropanoic acid hydrochloride (TD6)

¹H NMR (D₂O, 400 MHz) δ (ppm)=4.02 (br d, J=12.3 Hz, 2H), 3.73 (br t,J=12.3 Hz, 2H), 3.46 (br d, J=12.3 Hz, 2H), 3.39 (t, J=7.0 Hz, 2H), 3.13(br t, J=12.3 Hz, 2H), 2.79 (t, J=7.0 Hz, 2H).

5-morpholinopentanoic acid hydrochloride (TD12)

¹H NMR (D₂O, 400 MHz) δ (ppm)=4.02 (br d, J=12.3 Hz, 2H), 3.73 (br t,J=12.3 Hz, 2H), 3.46 (br d, J=12.3 Hz, 2H), 3.15-3.06 (m, 4H), 2.41 (t,J=7.1 Hz, 2H), 1.72-1.66 (m, 2H), 1.60-1.52 (m, 2H).

4-(piperidin-1-yl)butanoic acid hydrochloride (TD7)

¹H NMR (D₂O, 400 MHz) δ (ppm)=3.43 (br d, J=12.1 Hz, 2H), 3.04-2.99 (m,2H), 2.88 (td, J=2.7, 12.1 Hz, 2H), 2.38 (t, J=7.1 Hz, 2H), 1.95-1.80(m, 4H), 1.76-1.55 (m, 3H), 1.45-1.32 (m, 1H).

2-(piperidin-1-yl)acetic acid hydrochloride (TD8)

5-(piperidin-1-yl)pentanoic acid hydrochloride (TD9)

¹H NMR (D₂O, 400 MHz) δ (ppm)=3.43 (br d, J=12.1 Hz, 2H), 3.04-2.99 (m,2H), 2.80 (td, J=2.7, 12.1 Hz, 2H), 2.38 (t, J=7.1 Hz, 2H), 1.88-1.78(m, 2H), 1.76-1.49 (m, 7H), 1.45-1.32 (m, 1H).

3-(piperidin-1-yl)propanoic acid hydrochloride (TD13)

¹H NMR (D₂O, 400 MHz) δ (ppm)=3.43 (br d, J=12.1 Hz, 2H), 3.29 (t, J=7.1Hz, 2H), 2.88 (td, J=2.7, 12.1 Hz, 2H), 2.76 (t, J=7.1 Hz, 2H),1.85-1.80 (m, 2H), 1.76-1.62 (m, 3H), 1.45-1.32 (m, 1H).

2′-O-acylation (general procedure) TD4: Morpholino butanoic acid ester

A stirred solution of 4-morpholinobutanoic acid hydrochloride (0.095 g,0.45 mmol, 1.2 equiv) in pyridine (4 mL) and DBU (0.140 mL, 3 equiv) ina 25 mL round bottom flask was cooled in an ice bath at 0° C. andacetonitrile (2 mL) was added, followed by Taxotere® (0.303 g, 0.375mmol, 1 equiv). 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCI,0.180 g, 2.5 equiv) was added in portions over 15 minutes. The resultingsuspension was stirred, gradually warming to room temperature as the icebath melted over the course of 16 h. TLC analysis (30% Hexanes inEtOAc/6% MeOH (spiked with 5% NH₄OH) revealed almost complete conversionat this time. Ethanol (2 mL) was added and the flask was transferred toa rotary evaporator and concentrated at high vac. The resulting oil wasre-dissolved in ethanol and concentrated again. The dried residue wasdissolved in methylene chloride (˜4 mL) and loaded on to apre-equilibrated silica gel column (60 mL silica, 30% Hexanes inEtOAc/2% MeOH (spiked with 5% NH₄OH)) and eluted with increasingly polarsolvent mixtures (2-8% MeOH, 2% increments, 100 mL/increment). Fractionscontaining the desired material were pooled and concentrated to give thedesired compound (0.255 g, 71%).

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.71-7.48 (m,3H), 7.48-7.31 (m, 4H), 7.25 (m, 1H), 6.09 (m, 1H), 5.63 (d, J=7.1 Hz,1H), 5.40-5.16 (m, 3H), 4.99 (d, J=8.1 Hz, 1H), 4.28-4.13 (m, 3H), 3.86(br d, J=6.8 Hz, 1H), 3.64 (m, 4H), 2.67-2.10 (m, 14H), 1.99-1.72 (m,7H), 1.68 (s, 3H), 1.40 (s, 9H), 1.15 (s, 3H), 1.11 (s, 3H).

The material was recrystallized from EtOAc/Hexanes and used forbiological and solubility testing. After re-crystallization, HPLC/MS(m/z); 963.2 (m+H) 99.8% area by UV.

The same general procedure was used to generate the following analogs:

TD2: N-methyl-piperazinyl acetic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.66 (t, J=7.4Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 7.41 (m, 4H), 7.25 (m, 1H), 6.08 (m,1H), 5.62 (d, J=7.1 Hz, 1H), 5.42 (d, 2H), 5.27 (s, 1H), 4.99 (d, J=8.1Hz, 1H), 4.29-4.13 (m, 3H), 3.85 (br d, J=6.8 Hz, 1H), 3.42-3.33 (m,3H), 2.71-2.27 (m, 16H), 2.03 (q, 1H), 1.92 (s, 3H), 1.89-1.79 (m, 1H),1.68 (s, 3H), 1.39 (s, 9H), 1.15 (s, 3H), 1.11 (s, 3H). HPLC/MS (m/z);949.4 (m+H) 95% area by UV.

TD3: N-methyl-piperazinyl pentanoic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.66 (t, J=7.4Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 7.41 (m, 4H), 7.25 (m, 1H), 6.08 (m,1H), 5.62 (d, J=7.1 Hz, 1H), 5.37-5.18 (m, 3H), 4.99 (d, J=8.1 Hz, 1H),4.29-4.13 (m, 3H), 3.85 (d, J=6.8 Hz, 1H 1H), 3.63-3.48 (m, 5H),2.80-2.27 (m, 24H), 2.27-2.13 (m, 1H), 2.03 (q, 2H), 1.97-1.54 (m, 19H),1.57-1.49 (br m, H), 1.39 (s, 9H), 1.15 (s, 3H), 1.11 (s, 3H). HPLC/MS(m/z); 990.6 (m+H) 96% area by UV.

TD5: Morpholino acetic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.12 (d, J=7.4 Hz, 2H), 7.64 (t, J=7.4Hz, 1H), 7.59-7.50 (m, 2H), 7.44-7.34 (m, 4H), 7.32-7.20 (m, 1H), 6.16(m, 1H), 5.64 (d, J=7.1 Hz, 1H), 5.42 (br d, 2H), 5.27 (s, 1H), 5.01 (d,J=8.1 Hz, 1H), 4.27-4.15 (m, 3H), 3.89 (d, J=6.8 Hz, 1H), 3.71-3.58 (m,4H), 3.38 (d, 1H), 2.54-2.26 (m, 9H), 2.12-2.01 (m, 1H), 1.92 (s, 3H),1.87-1.76 (m, 1H), 1.69 (s, 3H), 1.39 (s, 9H), 1.15 (s, 3H), 1.11 (s,3H). HPLC/MS (m/z); 936.1 (m+H) 98% area by UV.

TD6: Morpholino propionic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.66 (t, J=7.4Hz, 1H), 7.61-7.50 (m, 2H), 7.45-7.33 (m, 4H), 7.30-7.20 (m, 1H), 6.09(m, 1H), 5.63 (d, J=7.1 Hz, 1H), 5.39-5.16 (m, 3H), 5.00 (d, J=8.1 Hz,1H), 4.27-4.13 (m, 3H), 3.86 (d, J=6.8 Hz, 1H), 3.70-3.54 (m, 4H),2.72-2.56 (m, 4H), 2.52-2.29 (m, 8H), 2.29-2.14 (m, 1H), 1.99-1.86 (m,4H), 1.86-1.75 (m, 2H), 1.68 (m, 13H), 1.39 (s, 9H), 1.15 (s, 3H), 1.11(s, 3H). HPLC/MS (m/z); 950.9 (m+H) 94% area by UV.

TD8: Piperidinyl acetic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.16 (d, J=7.4 Hz, 2H), 7.63 (t, J=7.4Hz, 1H), 7.54 (t, J=7.7 Hz, 2H), 7.44-7.34 (m, 4H), 7.27 (m, 1H), 6.17(m, 1H), 5.65 (d, J=7.1 Hz, 1H), 5.49-5.38 (m, 2H), 5.28 (s, 1H), 5.01(d, J=8.1 Hz, 1H), 4.24-4.15 (m, 3H), 3.90 (d, J=6.8 Hz, 1H), 3.36-3.32(m, 1H), 3.19-3.11 (m, 1H), 2.57-2.25 (m, 9H), 2.16-2.06 (m, 1H), 1.92(s, 3H), 1.87-1.76 (m, 1H), 1.69 (s, 3H), 1.65-1.50 (m, 4H), 1.40-1.22(m, 11H), 1.15 (s, 3H), 1.11 (s, 3H). HPLC/MS (m/z); 933.8 (m+H) 94%area by UV.

TD7: Piperidinyl butanoic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.65 (t, J=7.4Hz, 1H), 7.56 (t, J=7.7 Hz, 2H), 7.44-7.33 (m, 4H), 7.29-7.20 (m, 1H),6.09 (m, 1H), 5.63 (d, J=7.1 Hz, 1H), 5.36-5.30 (m, 1H), 5.29-5.26 (m,2H), 5.03-4.95 (m, 1H), 4.27-4.14 (m, 3H), 3.86 (d, J=6.8 Hz, 1H),2.59-2.16 (m, 15H), 2.01-1.75 (m, 8H), 1.68 (s, 3H), 1.64-1.54 (m, 5H),1.47 (m, 3H), 1.40 (s, 9H), 1.15 (s, 3H), 1.11 (s, 3H). HPLC/MS (m/z);962.5 (m+H) 94% area by UV.

TD9: Piperidinyl pentanoic acid ester

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.4 Hz, 2H), 7.66 (, J=7.4Hz, 1H), 7.57 (t, J=7.7 Hz, 2H), 7.45-7.33 (m, 4H), 7.25 (br s, 1H),6.08 (m, 1H), 5.62 (d, J=7.1 Hz, 1H), 5.31 (m, 1H), 5.28-5.18 (m, 2H),4.99 (d, J=8.1 Hz, 1H), 4.27-4.15 (m, 3H), 3.85 (d, J=6.8 Hz, 1H),2.51-2.18 (m, 17H), 1.97-1.75 (m, 5H), 1.75-1.46 (m, 20H), 1.43-1.38 (m,10H), 1.15 (s, 3H), 1.11 (s, 3H). HPLC/MS (m/z); 976.2 (m+H) 96% area byUV.

7-OH acylation (general procedure) Protection: GCW00006-09

To a stirred, cooled (−45° C.) solution of docetaxel (0.746 g, 0.923mmol) in methylene chloride (20 mL) and pyridine (1.6 mL) was addedtrichloroethyl chloroformate (Troc-Cl, 0.137 mL, 1.01 mmol, 1.1 equiv)in a dropwise fashion. The reaction was allowed to stir for 1 h atreduced temperature, and a second, equal portion of Troc-Cl (0.137 mL,1.01 mmol, 1.1 equiv) was added in a dropwise fashion. The reaction wasallowed to gradually warm to room temperature with stirring over thecourse of the next 16 h. At that time TLC analysis (30% EtOAc inHexanes) indicated a minimal amount of remaining starting material andthe formation of three new spots presumed to be the 2′,10-di-Troc, the2′,7-di-Troc and the 2′,7,10-tri-Troc protected compounds. The reactionwas diluted with a minimal amount of ethanol and concentrated to drynesson a high-vac equipped rotovap. The residue was then dissolved in aminimal amount of CH₂Cl₂ and loaded on to a pre-equilibrated column ofsilica gel (3 cm×20 cm, 20% EtOAc in hexanes). Careful elution of thedesired products from the column using increasingly polar solventmixtures (20-60% EtOAc in hexanes, 100 mL volumes, 5% increments), andcollection and concentration of the clean fractions yielded the desiredisomer as an amorphous white solid (0.433 g, 40%).

¹H NMR (400 MHz, CDCl₃) δ (ppm)=8.11 (d, 2H), 7.61 (t, 1H), 7.51 (t,2H), 7.45-7.33 (m, 5H), 6.29 (m, 1H), 6.16 (s, 1H), 5.69 (d, 1H),5.59-5.56 (m, 1H), 5.55-5.42 (m, 2H), 5.35 (br s, 1H), 4.96 (br d, 1H),4.89 (d, 1H), 4.76 (q, 2H), 4.69 (d, 1H), 4.41-4.37 (m, 1H), 4.32 (d,1H), 4.18 (d, 1H), 3.96-3.91 (m, 3H), 3.78 (d, 1H), 2.61-2.53 (m, 1H),2.43 (s, 3H), 2.39-2.18 (m, 3H), 2.11-1.76 (m, 11H), 1.73 (br s, 1H),1.69 (s, 3H), 1.32 (s, 9H), 1.28-1.17 (m, 6H).

The same general procedure was used to generate the following analogs:

Esterification: GCW00006-10

To a round bottom flask containing 4-(4-methylpiperazin-1-yl)butanoicacid hydrochloride (0.58 g, 2.61 mmol) and a magnetic stir bar was addedthionyl chloride (15 mL). The resulting solution was heated to refluxfor 1.5 h, cooled to room temperature, concentrated on a rotaryevaporator, suspended in anhydrous toluene (10 mL), concentrated on arotary evaporator again to yield a white solid and dried on a highvacuum line for 3 h to a steady weight that gave off no odor of thionylchloride or hydrochloric acid.

To a solution of GCW00006-09 (0.433 g, 0.375 mmol) in methylene chloride(Dri-Solve, 8 mL), containing a magnetic stirrer was addedN,N-dimethylamino-pyridine (DMAP, 0.229 g, 5 equiv). The solution wascooled to 0° C. and the above described amino acyl chloridehydrochloride (0.100 g, 1.1 equiv) was added in portions over the courseof a couple of minutes. The reaction was followed based on TLC analysisfor the consumption of starting material as the DMAP tended to co-elutewith the mono-amino acylated product. After 2 h, some remaining startingmaterial was still observed by TLC and an additional portion of theamino-acyl chloride hydrochloride was added (0.05 g, 0.55 equiv). Afteran additional hour with stirring at room temperature, TLC indicatedalmost complete consumption of the starting material. The reaction wasconcentrated on a rotary evaporator to give an oil that was dissolved ina minimal amount of CH₂Cl₂ (5 mL) and loaded on to a pre-equilibratedcolumn of silica (3 cm×20 cm, 4:1 CH₂Cl₂/Hexanes) and subjected to flashchromatography (4:1 CH₂Cl₂/Hexanes with 1-10% MeOH (containing 5%NH₄OH). Fractions containing the desired material were collected andconcentrated to give a colorless glass (0.284 g, 57%).

¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.11 (d, J=7.5 Hz, 2H), 7.61 (t, J=7.7Hz, 1H), 7.51 (t, J=7.7 Hz, 2H), 7.43-7.39 (m, 4H), 7.28-7.26 (m, 1H),6.13-6.05 (m, 2H), 5.64 (d, J=6.8 Hz, 1H), 5.60-5.56 (m, 1H), 5.55-5.42(m, 2H), 5.36-5.34 (m, 1H), 4.99 (d, J=6.8 Hz, 1H), 4.92 (d, J=11.2 Hz,1H), 4.83 (d, J=11.2 Hz, 1H), 4.19 (dd, J=8.2, 19.0 Hz, 1H), 3.85 (d,J=6.3 Hz, 1H), 2.67-2.22 (m, 20H), 2.05-1.89 (m, 4H), 1.82-1.71 (m, 6H),1.40 (s, 9H), 1.15 (s, 3H), 1.11 (s, 3H). HPLC/MS (m/z); 1325.7 (m+H)83% area by UV (contaminated with 10% methyl carbonate (m/z=1210.4)).

Deprotection to form TD10: 7-O-(N-methyl-piperazinyl butanoic acidester)

To a vigorously stirred solution of GCW00006-10 (0.276 g, 0.2 mmol) inmethanol and acetic acid (50 mL, 10% AcOH) was added elemental zinc dust(˜0.1 g). The reaction was monitored by TLC and within 1 h all of thestarting material had been consumed and converted to a singular lowerrunning spot (10% MeOH (w 5% NH₄OH) in CH₂Cl₂). The reaction was dilutedwith MeOH (50 mL) and filtered on a filter paper lined Buchner funnel.The resulting solution was concentrated to dryness on a rotaryevaporator to give a stiff syrup that was dissolved in CH₂Cl₂ (5 mL) andloaded on to a pre-equilibrated column of silica gel (3 cm×15 cm, 2%MeOH (w 5% NH₄OH) in CH₂Cl₂) and eluted with increasing polar solvent(2-10% MeOH (w 5% NH₄OH) in CH₂Cl₂, 2% increments, 150 mL/increment).Fractions containing the clean material, as determined by TLC werecollected and concentrated to give a white solid (0.0764, 39%). HPLC/MSindicates minor contamination (˜10%) containing a methyl carbonatesubstituent at undetermined location on the parent compound.

(m/z=1035.3) ¹H NMR (400 MHz, CD₃OD) δ (ppm)=8.12 (d, J=7.5 Hz, 2H),7.68 (t, J=7.7 Hz, 1H), 7.58 (t, J=7.7 Hz, 2H), 7.43-7.39 (m, 4H),7.28-7.26 (m, 1H), 6.13-6.05 (m, 2H), 5.67 (d, J=6.8 Hz, 1H), 5.60-5.56(m, 1H), 5.38 (s, 1H), 5.14 (br s, 1H), 5.01 (d, J=6.8 Hz, 1H), 4.52 (brs, 1H), 3.98 (d, J=6.3 Hz, 1H), 2.67-2.22 (m, 18H), 2.10-1.71 (m, 10H),1.40 (s, 9H), 1.15 (s, 3H), 1.12 (s, 3H). ¹³C NMR (100 MHz, CD₃OD) δ(ppm)=209.32, 173.04, 172.30, 170.61, 166.25, 156.36, 145.92, 139.27,138.27, 136.40, 133.20, 129.98, 129.78, 128.31, 128.19, 127.38, 126.83,83.70, 80.46, 79.32, 77.75, 76.12, 74.86, 74.24, 74.03, 71.68, 71.00,57.16, 57.00, 56.14, 54.18, 52.14, 46.06, 44.51, 42.98, 37.97, 35.41,32.98, 31.33, 31.19, 27.30, 25.39, 21.69, 21.43, 21.28, 20.17. HPLC/MS(m/z); 977.1 (m+H) 85% area by UV.

Example 3 Water-Soluble Prednisone DerivativesN-methyl-piperazinyl-butanoic acid ester

Linker Synthesis

A mixture of ethyl 4-bromobutanoate (5.75 g, 29.5 mmol; Aldrich No.167118) and 1-methylpiperazine (3.55 mL, 32.0 mmol; Aldrich No. 130001)and anhydrous K₂CO₃ (4.5 g, 32.5 mmol; Fisher No. P208) in acetonitrile(MeCN, 150 mL) was refluxed for 18 h before concentrated in vacuo. Theorganic layer was then separated and the aqueous layer was extractedwith dichloromethane (DCM, 3×150 mL). The combined organic extracts werewashed with water (150 mL), dried (Na₂SO₄), and concentrated in vacuo togive ethyl 4-(4-methylpiperazin-1-yl)butanoate (6.01 g, 96%) as a yellowoil.

¹H NMR (CDCl₃): 4.03 (q, 2H, J=7.15 Hz), 2.29-2.44 (m, 7H), 2.21-2.28(m, 5H), 2.18 (s, 3H), 1.67-1.75 (m, 2H), 1.18 (t, 3H, J=7.14 Hz)

¹³C NMR (CDCl₃): 174.4, 61.1, 58.5, 56.1, 54.0, 47.0, 33.2, 23.1, 15.2

ESI-MS: 215.1 [M+H]⁺; 237.2 [M+Na]⁺

To a solution of ethyl 4-4(methylpiperazin-1-yl)butanoate (6.01 g, 28.1mmol) in tetrahydrofuran (THF, 150 mL) was added a solution of NaOH(1.20 g, 30 mmol) in water (150 mL). The mixture was stirred at RT for18 h before concentrated to dryness to give sodium4-(4-methylpiperazin-1-yl)butanoate (6.06 g, quant.) as a white powder.

¹³C NMR (MeOH-d₄): 181.0, 58.2, 54.2, 52.4, 44.8, 35.7, 23.2

ESI-MS: 187.3 [M+H]⁺; 209.2 [M+Na]⁺

Esterification

To a suspension of sodium 4-(4-methylpiperazin-1-yl)butanoate (128 mg,0.615 mmol) and prednisone (200 mg, 0.559 mmol) in acetonitrile (MeCN,10 mL) was added 2-chloro-1-methyl-pyridinium iodide (235 mg, 0.922mmol; Aldrich No. 198005). The resulting suspension was stirred at RTfor 18 h before quenching with water (30 mL). The product was thenextracted with ethylacetate (EtOAc, 4×20 mL), washed with sat. aq.NaHCO₃ (3×20 mL) and brine (20 mL), dried (Na₂SO₄), and concentrated invacuo. Further purification was performed on a silica gel column(solvent: 1% NH₄OH, 10% MeOH, 89% dichloromethane) to give the free baseof the derivatized prednisone (108 mg, 36%) as a white solid.

¹H NMR (CDCl₃): 7.64 (d, 1H, J=10.40), 6.08 (dd, 1H, J=10.40, 1.90),6.06 (t, 1H, J=2.90), 5.06 (ABq, 2H, J=94.78, 17.81), 2.85 (d, 1H,J=12.32), 2.65 (t, 1H, J=12.57), 2.56-1.06 (CM, 32H), 2.16 (s, 3H), 1.34(s, 3H), 0.57 (s, 3H)

¹³C NMR (CDCl₃): 209.17, 205.05, 186.61, 172.86, 167.44, 155.93, 127.29,124.31, 88.18, 67.78, 60.05, 57.30, 54.92, 52.73, 51.28, 49.97, 49.56,45.89, 42.47, 36.00, 34.47, 33.64, 32.23, 31.63, 23.21, 21.93, 18.68,15.30

MS: 527.4 [M+H]⁺

N-methyl-piperazinyl acetic acid ester Linker Synthesis

A mixture of ethyl 2-bromoacetate (4.93 g, 29.5 mmol),1-methylpiperazine (3.55 mL, 32.0 mmol; Aldrich No. 130001), and K₂CO₃(4.5 g, 32.5 mmol; Fisher No. P208) in CH₃CN (150 mL) was refluxed for18 h before concentrated in vacuo. The organic layer was then separatedand the aqueous layer was extracted with DCM (3×150 mL). The combinedorganic extracts were washed with water (150 mL), dried (Na₂SO₄), andconcentrated in vacuo to give ethyl 4-(4-methylpiperazin-1-yl)acetate(5.26 g, 96%) as a yellow oil.

¹H NMR (CDCl₃): 3.76 (q, 2H, J=7.14 Hz), 2.76 (s, 3H), 2.30-1.90 (br,4H), 0.85 (t, 3H, J=7.14 Hz)

¹³C NMR (CDCl₃): 169.55, 59.90, 58.93, 54.44, 53.26, 52.47, 45.59, 13.84

ESI-MS: 187 [M+H]⁺

To a solution of ethyl 2-4(methylpiperazin-1-yl)acetate (5.26 g, 28.3mmol) in THF (150 mL) was added a solution of NaOH (1.20 g, 30 mmol) inwater (150 mL). The mixture was stirred at RT for 18 h beforeconcentrated to dryness to give sodium 4-(4-methylpiperazin-1-yl)acetate(5.24 g, quant.) as a white powder.

¹³C NMR (MeOH-d₄): 169.55, 59.90, 58.93, 54.44, 52.47, 45.59

ESI-MS: 187.3 [M+H]⁺; 209.2 [M+Na]⁺

Esterification

To a suspension of sodium 2-(4-methylpiperazin-1-yl)acetate (111 mg,0.615 mmol) and prednisone (200 mg, 0.559 mmol) in CH₃CN (10 mL) wasadded 2-chloro-1-methyl-pyridinium iodide (235 mg, 0.922 mmol; AldrichNo. 198005). The resulting suspension was stirred at RT for 18 h beforequenching with water (30 mL). The product was then extracted with EtOAc(4×20 mL), washed with sat. aq. NaHCO₃ (3×20 mL) and brine (20 mL),dried (Na₂SO₄), and concentrated in vacuo. Further purification wasperformed on a silica column (solvent: 1% NH₄OH, 10% MeOH, 89% DCM) togive the derivatized prednisone (154 mg, 38%) as a white solid.

¹H NMR (CDCl₃): 7.71 (d, 1H, J=10.28), 6.19 (dd, 1H, J=10.24, 1.96),6.07 (t, 1H, J=1.93), 4.93 (ABq, 2H, J=124.45, 17.56), 3.33 (s, 1H),2.89 (d, 1H, J=13.36), 2.84-1.17 (CM, 32H), 2.27 (s, 3H), 1.43 (s, 3H),0.66 (s, 3H)

¹³C NMR (CDCl₃): 208.88, 204.58, 186.58, 169.85, 167.06, 155.68, 127.49,124.50, 88.38, 67.96, 60.22, 59.01, 54.73, 53.42, 52.71, 51.43, 49.67,49.56, 46.00, 42.45, 36.06, 34.79, 33.73, 32.25, 23.26, 18.75, 15.45

MS: 449.3 [M+H]⁺

Example 4 Lipophilic Prednisone Derivatives Internal Linoleyl Linkers1-(tert-butyldimethylsilyloxy)-3-(dimethylamino)propan-2-ol

A dry dichloromethane (10 mL) solution of3-(dimethylamino)-1,2-propanediol (98%, 1.00 g, 8.39 mmol, 1.0 equiv)and imidazole (0.57 g, 8.39 mmol, 1.0 equiv) was stirred at 0° C. underargon for 15 minutes. Solid tert-butyldimethylsilyl chloride (1.26 g,8.39 mmol, 1.0 equiv) was added to the mixture and the resultant wasstirred for 2 hours at 0° C. The mixture was then diluted with 20 mL ofdichloromethane and poured into deionized water (15 mL). The organiclayer was separated and the aqueous layer was extracted with twoadditional portions of dichloromethane (20 mL). The combined organicextracts were dried (MgSO₄), filtered and concentrated to afford crude1-(tert-butyldimethylsilyloxy)-3-(dimethylamino)propan-2-ol, a thickclear oil, which was used without further purification.

¹H NMR: 3.72-3.80 (m, 1H), 3.63 (d, 2H, J=5.19), 2.34-2.46 (m, 2H), 2.33(s, 6H), 0.91 (2, 9H), 0.08 (s, 6H)

3-(tert-butyldimethylsilyloxy)-N,N-dimethyl-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-amine

A toluene (10 mL) solution of crude1-(tert-butyldimethylsilyloxy)-3-(dimethylamino)propan-2-ol (1.0 g, 4.29mmol, 1.0 equiv) was carefully added dropwise to a toluene suspension (5mL) of NaH (60%, 0.17 g, 4.29 mmol, 1.0 equiv) at 0° C. under argon andthe resultant was stirred for 15 minutes. A toluene solution (5 mL) oflinoleyl methanesulfonate (1.47 g, 4.29 mmol, 1.0 equiv) was addeddropwise to the stirring mixture and the reaction was then stirred for18 hours at 90° C. The mixture was then cooled to room temperature andquenched by the slow addition of ethanol (10 mL). The mixture was thenconcentrated and the residue was taken up with deionized water (15 mL)and extracted three times with EtOAc (20 mL). The combined organicextracts were washed with deionized water (15 mL), dried (MgSO₄),filtered, and concentrated. Chromatographic purification of the residue(0-5% MeOH in chloroform) yielded 109 mg (53% yield) of3-(tert-butyldimethylsilyloxy)-N,N-dimethyl-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-amine,a thick clear oil.

¹H NMR: 5.28-5.42 (m, 4H), 3.47-3.62 (m, 4H), 3.37-3.42 (m, 1H), 2.77(t, 2H, J=5.94), 2.28-2.47 (m, 2H), 2.25 (s, 6H), 2.01-2.09 (m, 5H),1.50-1.58 (m, 2H), 1.30 (br, 18H), 0.09 (s, 9H), 0.06 (s, 6H).

¹³C NMR: 130.22, 130.01, 127.98, 127.88, 70.16, 63.07, 37.35, 32.79,31.52, 29.59, 29.49, 29.39, 29.34, 29.32, 29.23, 29.15, 29.11, 29.00,27.19, 25.72, 25.62, 25.40, 22.57, 14.07.

3-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-ol

TBAF (1.0 M in THF, 0.5 mL, 0.50 mmol, 1.2 equiv) was added in oneportion to a dry THF (100 μL) solution of3-(tert-butyldimethylsilyloxy)-N,N-dimethyl-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-amine(0.2 g, 0.42 mmol, 1.0 equiv) and the mixture was stirred at roomtemperature for 2 hours. The mixture was concentrated and the residuewas partitioned between EtOAc (15 mL) and aqueous saturated ammoniumchloride solution (10 mL). The layers were separated and the aqueouslayer was extracted with 2 additional portions of EtOAc (10 mL). Thecombined extracts were dried (MgSO₄), filtered, and concentrated to givecrude 3-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-ol,a thick, beige oil, which was used without further purification.

¹H NMR: 5.29-5.43 (m, 4H), 3.77-3.82 (m, 1H), 3.65-3.71 (m, 1H),3.42-3.51 (m, 3H), 2.78 (t, 2H, J=5.97), 2.54-2.57 (m, 2H), 2.30 (s,6H), 2.02-2.09 (m, 4H), 1.50-1.57 (m, 2H), 1.30 (br, 15H), 0.87-0.92 (m,4H).

4-(3-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propoxy)-4-oxobutanoicacid

Succinic anhydride (0.60 g, 5.99 mmol, 1.1 equiv) was added in oneportion to a dry THF (11 mL) solution of3-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propan-1-ol (2.0g, 5.45 mmol, 1.0 equiv) and the resultant was refluxed for 18 hoursunder argon. The mixture was concentrated and then dissolved in EtOAc(10 mL) and poured into deionized water (20 mL). The layers wereseparated and the aqueous layer was extracted with two additionalportions of EtOAc (25 mL). The combined organic extracts were dried(MgSO₄), filtered and concentrated to provide crude4-(3-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propoxy)-4-oxobutanoicacid, a thick yellow oil, which was used without further purification.

¹H NMR: 5.29-5.43 (m, 4H), 4.28 (d, 1H, J=11.25), 3.92-3.98 (m, 1H),3.84 (br, 1H), 3.60-3.67 (m, 1H), 3.43-3.50 (m, 1H), 3.11-3.15 (d, 1H,J=12.45), 2.75-2.79 (m, 2H), 2.67 (s, 6H), 2.53-2.65 (m, 5H), 2.01-2.08(m, 4H), 1.53-1.57 (m, 2H), 1.29-1.30 (br, 18H), 0.87-0.91 (m, 3H).

¹³C NMR: 176.63, 172.55, 130.09, 129.98, 127.91, 127.84, 73.90, 69.90,67.85, 62.54, 59.08, 44.11, 31.44, 30.53, 29.97, 29.93, 29.58, 29.42,29.36, 29.27, 29.20, 27.14, 27.11, 26.04, 25.55, 22.50, 14.01.

External Linoleyl Linkers1-(dimethylamino)-4-((9Z,12Z)-octadeca-9,12-dienyloxy)butan-2-ol

A toluene (10 mL) solution of 3-(dimethylamino)-1,2-propanediol (98%,1.00 g, 8.39 mmol, 1.0 equiv)) was carefully added dropwise to a toluenesuspension (5 mL) of NaH (60%, 0.34 g, 8.39 mmol, 1.0 equiv) at 0° C.under argon and the resultant was stirred for 15 minutes. A toluenesolution (5 mL) of linoleyl methanesulfonate (2.87 g, 8.39 mmol, 1.0equiv) was added dropwise to the stirring mixture and the reaction wasthen stirred for 18 hours at 90° C. The mixture was then cooled to roomtemperature and quenched by the slow addition of ethanol (10 mL). Themixture was concentrated and the residue was taken up with deionizedwater (20 mL) and extracted three times with EtOAc (30 mL). The combinedorganic extracts were washed with deionized water (20 mL), dried(MgSO₄), filtered, and concentrated. Chromatographic purification of theresidue (0-5% MeOH in chloroform) provided1-(dimethylamino)-4-((9Z,12Z)-octadeca-9,12-dienyloxy)butan-2-ol, athick, clear oil.

4-(1-(dimethylamino)-4-((9Z,12Z)-octadeca-9,12-dienyloxy)butan-2-yloxy)-4-oxobutanoicacid

Succinic anhydride (0.60 g, 5.99 mmol, 1.1 equiv) was added in oneportion to a dry THF (11 mL) solution of1-(dimethylamino)-4-((9Z,12Z)-octadeca-9,12-dienyloxy)butan-2-ol (2.0 g,5.45 mmol, 1.0 equiv) and the resultant was refluxed for 18 hours underargon. The mixture was concentrated and the residue was partitionedbetween EtOAc (10 mL) and deionized water (20 mL). The layers wereseparated and the aqueous layer was extracted with two additionalportions of EtOAc (25 mL). The combined organic extracts were dried(MgSO₄), filtered and concentrated to provide crude4-(1-(dimethylamino)-4-((9Z,12Z)-octadeca-9,12-dienyloxy)butan-2-yloxy)-4-oxobutanoicacid, a thick, yellow oil, which was used without further purification.

Esterification of Prednisone with Internal Linker

NEt₃ (100 μL, 0.74 mmol, 1.0 equiv) was added dropwise to a drydichloromethane solution (10 mL) of4-(3-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dienyloxy)propoxy)-4-oxobutanoicacid and the resultant was stirred for 15 minutes under argon at roomtemperature. PyBOP (0.48 g, 0.93 mmol, 1.25 equiv) was added in oneportion and the resultant was stirred for 10 minutes. Prednisone (0.32g, 0.89 mmol, 1.2 equiv) was added to the mixture and the resultant wasstirred at room temperature for 18 hours and then concentrated toprovide a thick yellow oil, (90% pure). Purification by flash columnchromatography (5-15% MeOH in chloroform and (0-15% MeOH in EtOAc)provided the desired product

¹H NMR (CDCl₃): 7.68 (d, 1H, J=10.23), 6.20 (dd, 1H, J₁=10.23, J₂=1.86),6.07 (s, 1H), 5.27-5.42 (m, 4H), 4.80 (dd, 1H, J₁=17.70, J₂=5.61),4.09-4.36 (m, 3H), 3.54-3.69 (m, 3H), 3.34 (m, 1H), 3.07-3.22 (m, 1H),2.94-2.98 (m, 1H), 2.88 (s, 6H), 2.74-2.78 (m, 3H), 2.65-2.68 (m, 3H),2.37-2.50 (m, 3H), 2.25 (dd, 1H, J₁=12.24, J₁=2.31), 2.00-2.05 (m, 8H),1.42 (s, 3H), 1.30 (br, 18H), 0.89 (m, 3H), 0.64 (s, 3H).

MS: 808.8 [M+H]⁺.

Example 5 Etoposide Derivatives

4-(4-Methylpiperazin-1-yl)butanoic acid dihydrochloride salt (4, 20 mg,0.09 mmol) was dissolved in SOCl₂ (0.5 mL), and stirred under argonatmosphere at room temperature for 3 h. SOCl₂ was evaporated, andwithout further purification the crude acid chloride was dissolved indry CH₃CN (1 mL) under argon atmosphere. The solution was cooled to 0°C., etoposide (6, 50 mg, 0.085 mmoles) dissolved in CH₃CN (1 mL) wasadded dropwise, followed by triethylamine (10 μl). Stirring wascontinued for 2 h, with monitoring of the reaction by TLC. The solutionwas then concentrated in vacuo and the crude product was taken up withwater and extracted with ethyl acetate (3×10 mL). The combined organicextracts were dried over anhydrous sodium sulfate and concentrated invacuo. The crude product was purified by silica gel (230-400 mesh)column chromatography (gradient 5-10% MeOH in CH₂Cl₂) to give 30 mg ofdesired free base of etoposide derivative 7 as a white solid.

¹H NMR (CDCl₃): 6.83 (s, 1H), 6.55 (s, 1H), 6.27 (s, 2H), 5.98-6.00 (d,2H, J=7.15 Hz), 4.91-4.90 (d, 2H), 4.73-4.78 (q, 1H), 4.63-4.67 (t, 2H),4.40-4.46 (t, 1H), 4.15-4.26 (m, 2H), 3.75-3.78 (t, 2H), 3.66 (s, 3H),3.55-3.62 (m), 3.49 (s, 3H), 3.25-3.47 (m), 2.81-2.93 (m), 2.44-2.64(m), 2.31 (s, 3H), 1.87-1.97 (m), 1.39-1.40 (d, 3H).

ESI-MS: 757.5 [M+H]⁺.

Example 6 Tacrolimus Derivatives

4-(4-Methylpiperazin-1-yl)butanoic acid hydrochloride salt (4, 25 mg,0.12 mmol) was dissolved in SOCl₂ (0.5 mL), and stirred under argonatmosphere at room temperature for 3 h., then SOCl₂ was evaporated andwithout further purification compound 5 was dissolved in dry CH₃CN (1mL) under argon atmosphere. The solution was cooled to 0° C., thentacrolimus (8, 80 mg, 0.1 mmoles) dissolved in CH₃CN (1 mL) was added,followed by triethylamine (10 μL), Stirring was continued for 2 h, thenthe solution was concentrated in vacuo. The crude product was taken upwith water and extracted with ethyl acetate (3×15 mL). The combinedorganic extracts were dried over anhydrous sodium sulfate andconcentrated in vacuo. The crude product was purified by silica gel(230-400 mesh) column chromatography (gradient 5-10% MeOH in CH₂Cl₂) togive 45 mg of desired free base of tacrolimus derivative 9 as a whitesolid.

¹H NMR (CDCl₃): 5.63 (m, 1H), 5.20-5.32 (m, 2H), 6.27 (s, 2H), 4.98-5.10(m), 4.80 (d, 1H), 4.63-4.63 (d, 1H), 4.41-4.46 (d, 1H), 4.25 (s),3.87-3.92 (m, 2H), 3.67-3.75 (m), 3.56-3.60 (m), 3.30-3.44 (m),2.97-3.06 (br m), 2.30-2.75 (br m), 0.81-2.28 (broad continuousmultiplets) (spectra attached)

ESI-MS: 973.0 [M+H]⁺.

Example 7 Cyclosporine and Azathioprine Derivatives

Cyclosporine and azathioprine derivatives, such as the derivatives shownbelow, will be prepared essentially according to the method describedfor tacrolimus involving reaction of the parent drug with an acidchloride. It is well established that azathioprine reacts selectively atN-9 with electrophilic agents (Mishra et al., Ind. J. Chem., Sec. B,26B:847-50 (1987)). Thus, in some aspects, azathioprine derivativesprovided herein will be derivatized at N-9.

Example 8 Solubility of Weak-Base Derivatives

The solubility of the docetaxel derivatives was determined in acetatebuffer at pH 5, which is the buffer used for active loading into LN.Compounds were dissolved in ethanol at 50 mg/ml (except TD2, which wasdissolved at 25 mg/ml)). An aliquot was diluted 10-fold with 10 mMacetate buffer (pH 5) and the pH was checked and re-adjusted asnecessary to reach pH 5. Alternatively, 10 mg of each compound wasweighed into a glass vial, and 2 mL of 10 mM acetate buffer (pH 5) wasadded to the compound, followed by sonication of the suspension for 10minutes. The precipitate was then removed using Microcon MY100 filters(MW cut-off 100,000 Da) and the filtrate analyzed by UPLC-UV for drugcontent. The measured solubilities are kinetic solubilities determinedunder non-equilibrium conditions.

Small (non membrane-permeabilizing) quantities of ethanol can optionallybe used to increase aqueous solubility during loading. Thus, solubilitydata were generated in both buffer and buffer containing 10% (v/v)ethanol. The aqueous solubility of docetaxel derivatives (Table 2)varied significantly, with values ranging from about 20-500 timesgreater than that of docetaxel (Du et al., Bioorganic & MedicinalChemistry, 15:6323-30 (2007)). Solubility decreased in the orderN-methyl piperazino>piperidino>>morpholino. The solubility of themorpholino derivatives was significantly lower than that of thepiperazino and piperidino derivatives.

TABLE 2 Aqueous solubility of docetaxel derivatives at pH 5 in theabsence and presence of 10% ethanol. Solub. 10% EtOH Solub. in 10 mMacetate Prodrug (mg/ml) pH 5 (mg/ml) TD1 3.5 1.7 TD2 2.5 2 TD3 4.1 2.5TD4 — 0.12 TD5 0.3 0.14 TD6 1.5 0.48 TD7 — 1.9 TD8 — 0.5 TD9 2.3 1 TD10— 3

The pKa of the amino group of TD1 was determined by acid-base titrationto be 7.7, making it well suited for pH gradient loading into LN. Asexpected, the water solubility of the TD1 hydrochloride salt decreasedwith increasing pH (2.8 mg/ml at pH 4 and 1.7 mg/ml at pH 5).

Example 9 Stability of Weak-Base Derivatives

The chemical stability of the docetaxel derivatives was determined inaqueous solutions at different pH values and temperatures, and inbiological media (mouse plasma). Aliquots of the docetaxel derivativesin acetonitrile were mixed with buffered citrate/HEPES (10 mM/10 mM)solutions at pH 4.0 and 7.5, or in mouse plasma in 1 mL glass HPLCsample vials sealed with Teflon-lined caps (final volume 0.25 ml, finaldocetaxel derivative concentration 50 μg/ml). Drug stability wasdetermined 1, 4 and 24 hrs after incubation at 37° C. by UPLC-UV. At theindicated time points, a 3-fold excess of methanol/0.1% TFA was added tothe sample. Citrate/HEPES buffered samples were analyzed by UHPLC asdescribed above. For plasma samples, proteins precipitated by theaddition of methanol/0.1% TFA pelleted by centrifugation at 14,000×g andsupernatants were analyzed for the drug derivatives. Heparanized mouseplasma was diluted to 50% with 100 mM sodium-phosphate buffer to keepthe pH constant throughout the experiment.

To be suitable for formulation in LN, derivatives must be stable at pH 4(the pH present inside the LN carrier). In addition, prodrug derivativesshould readily form the active drug under physiological conditions(e.g., at pH 7.4 and/or in the presence of endogenous enzymes) oncereleased from the LN. Table 3 shows the hydrolytic stability ofdocetaxel derivatives at pH 4, pH 7.4, and in mouse plasma after 24hours of incubation at 37° C. Among the C-2′ amino ester docetaxelderivatives, TD1-4, TD7 and TD9 had adequate stability at pH 4. TD4 hasextremely low solubility in water and incubation in plasma appeared tohave no effect on TD9. TD 1-3 and TD7 were selected for loading into LNand testing of such LN for drug release in vitro.

The results (Table 3 and FIG. 1) indicate that the derivatives arestable at the low pH values found inside LN (pH around 4) and arecapable of undergoing rapid conversion into active drug followingrelease from the LN carrier in vivo. The conversion to active drug ispH-dependent (faster at higher pH) and is significantly accelerated inthe presence of hydrolytic enzymes present in biological fluids such asblood plasma.

TABLE 3 Prodrug levels (% remaining) after 24 hour incubation in pH 4 or7.4 buffer or mouse plasma. TD-1 TD-2 TD-3 TD-4 TD-5 TD-6 TD-7 TD-8 TD-9pH 4 94 99 99 98 81 68 94 78 99 pH 7.4 23 59 69 92 64 18 13 36 65 Plasma12 31 29 64 45 0 8 4 60

Example 10 Loading Efficiency

Piperazinyl ester (TD1-TD3), piperidine ester (TD7), and C-7 amino ester(TD10) derivatives of docetaxel, the N-methyl-piperazinyl butanoic acidand acetic acid ester derivatives of prednisone and theN-methyl-piperazinyl butanoic acid ester derivative of etoposide weretested for efficiency of loading into LN.

Preparation of LN

LN were prepared based on the ethanol procedure described by Boman etal., Cancer Res.; 54:2830-2833 (1994). Briefly, lipids(phospholipid/Chol, 55/45 molar ratio) were dissolved in ethanol andadded slowly into an aqueous solution containing 350 mM ammonium sulfateat 60° C.; trace amounts of the lipid marker [3^(H)]CHE (0.15 μCi/mgtotal lipid) were co-dissolved with the other lipids in ethanol toprepare LN for release studies. The final ethanol concentration was 15%(v/v). The resulting LN dispersions were extruded at 60° C. through twostacked 100 nm polycarbonate filters (Nucleopore, Pleasanton, Calif.)using a heated thermobarrel extruder (Northern Lipids, Vancouver,Canada), as described by Hope et al., Biochim. Biophys. Acta; 812: 55-65(1985). Residual ethanol and external ammonium sulfate were removed bytangential flow diafiltration at room temperature, and replaced with a300 mM sucrose solution using a Midgee™ HOOP™ ultrafiltration cartridge(MW cutoff 100000; Amersham Biosciences). Quasi-elastic light scattering(QELS) was used to assess the size distribution of the extruded LN(target size 100±20 nm), using a NICOMP model 370 submicron particlesizer (Particle Sizing Systems, Santa Barbara, Calif.).

Drug Loading

The docetaxel, etoposide and prednisone derivatives were loaded into

DSPC/Chol (55:45 mol %) LN using the ammonium sulfate-basedremote-loading method described by Haran et al., Biochim. Biophys. Acta;1151:201-15 (1993). Briefly, TD1 was dissolved at 2 mg/mL in 10 mMsodium acetate-buffered 300 mM sucrose (pH 5), the etoposide derivativewas dissolved at 2.5 mg/ml 10 mM sodium acetate-buffered 300 mM sucrose(pH 5) and the prednisone derivative was dissolved at 7 mg/mL in 10 mMsodium acetate buffer (pH 5.3). The dissolved derivatives were added topre-heated (60° C.) LN suspensions and the mixtures were incubated withstirring at 60° C. for the indicated times (typically 30 min). LNformulations are typically prepared at lipid concentrations between 5-10mg/ml and drug-to-lipid weight ratios of 0.1-0.4 mg/mg. Theunencapsulated docetaxel derivatives were removed by tangential flowdiafiltration using a Midgee™ HOOP™ ultrafiltration cartridge (MW cutoff100000; Amersham Biosciences) or size exclusion chromatography. Theexternal solution was replaced with non-buffered physiological salinesolution and the sample concentrated as needed. Drug-loaded LNformulations for in vivo studies were sterilized by filtration through0.2 μm filters (Nalgene) and subsequently stored at 4° C. TD-1 was alsoloaded into LN composed of DPPC/chol (55:45 mol %) and DMPC/chol (55:45mol %).

Loading efficiencies were determined by quantitating both prodrug andlipid levels before and after separation of external (non-encapsulated)prodrug from LN encapsulated prodrug by size exclusion chromatographyusing Sephadex G50 spin columns and comparing the respectiveprodrug/lipid ratios. Phospholipid concentrations were determined by thephosphorus assay of Fiske and Subbarow, J. Biol. Chem.; 66: 375-379(1925), and cholesterol concentrations were quantitated using anenzymatic colorimetric assay (Wako Chemicals, Richmond, Va.). Derivativeconcentrations were determined by ultra high performance liquidchromatography (UHPLC) as described herein. Conversion of the prodruginto parent drug during loading was monitored as well. Results are shownin FIGS. 2 (docetaxel derivatives), 3 (prednisone derivatives) and 4(etoposide derivative).

Example 11 Stability of LN Formulations

LN-derivative formulations with different lipid compositions (DSPC/Chol,DPPC/Chol and DMPC/Chol, each at 55/45 mol % and a derivative/lipidratio of 0.2 wt/wt) were prepared at a derivative concentration of 3mg/ml in 0.9% physiological saline. The LN formulations were sterilefiltered and sterile-filled into 5 mL glass vials and the vials werestoppered and capped and then stored at 7° C. At various time points(once a week within the first month and monthly thereafter) over a 4month period, formulations were analyzed for LN size (QELS), derivativeretention (Sephadex G-50 spin column method) and derivative integrity.Results are summarized in FIG. 5A-C. All three formulations wereextremely stable; prodrug release was not detectable (FIG. 5B); theaverage size and size distribution of the LN formulations remainedunchanged (FIG. 5C); and prodrug hydrolysis was less than 4% (3.5-3.8%,FIG. 5A). No other degradation products were observed. The datademonstrates the feasibility of developing wet LN formulations. Freezingof formulations is another alternative.

LN prepared using an ammonium sulfate gradient technique have anintravesicular pH of approximately 4.0 (Maurer-Spurej et al., Biochim.Biophys. Acta, 1416:1-10 (1999)). In light of the significantly greaterstability of the derivatives at pH 4 relative to pH 7.4 (see Table 2,above), LN encapsulation greatly improves the hydrolytic stability ofthe entrapped derivatives compared with the derivatives in aqueoussolution. For example, at pH 4 TD 1 has a hydrolysis half-life of about49 days (or 7 weeks). In contrast, less than 3% of the encapsulated TD1was converted into docetaxel over a period of 4 months (16 weeks).Cryo-TEM microscopy revealed that the prodrug is precipitated in the LNinterior and as a consequence has significantly higher stability.

The rate at which TD1 (formulated in the same manner as Taxotere™ and inLN) and Taxotere™ are removed from the blood circulation wasinvestigated following i.v. administration of the formulations to SwissWebster mice. The mice were injected intravenously with a single bolusinjection of equimolar doses (20 mg/kg docetaxel) of the formulations,and plasma levels of TD1 and docetaxel were determined by UHPLC-MS.Results are shown in FIG. 5. Both docetaxel/Taxotere™ and the derivativehad plasma circulation half-lives of minutes and plasma concentrationsbelow detectable levels by 2 h (FIG. 6). In contrast, formulation of thederivative in DSPC/Chol LN extended the circulation half-life fromminutes to 10-12 hours with two orders of magnitude higher plasmaconcentrations (FIG. 6). Approximately 24% of the injected dose remainedin the circulation at 16 h. The elimination of the LN-formulatedderivative appears to be primarily determined by the elimination rate ofthe LN carrier. The data demonstrate that LN formulations of thederivative are stable in circulation and can achieve circulationhalf-lives that favor efficient drug accumulation at therapeutictargets.

Example 12 Release of Drug Derivatives from LN In Vitro

The activity of LN-based drugs is highly dependent on the release rateof the drug from the carrier. For example, if the drug rapidly leaks outof the LN carrier, LN reaching the disease site will carry little or nodrug and there will be negligible therapeutic benefit over the freedrug. On the other hand, if a drug is released too slowly from the LN,the amount of drug reaching the disease site will never reachtherapeutic concentrations. The main determinants of drugretention/release are the lipid composition of the LN carrier and theintra-vesicular form of the drug. The use of unsaturated lipids orlipids with shorter acyl chains favours faster drug release. Drugprecipitation inside the LN can increase drug retention. Whether drugderivatives precipitate within LN can be determined by viewing LNformulations using cryo-TEM and/or other methods known in the art.

In vitro release of docetaxel derivatives from LN was assessed in mouseplasma. Drug retention was determined by comparison of the initialprodrug-to-lipid ratio with the prodrug-to-lipid ratios found atdifferent time points. Docetaxel derivatives were encapsulated inDSPC/chol, DPPC/Chol and DMPC/Chol LN (55:45 mol %) containing traceamounts of the radiolabeled lipid marker ³H-cholesterylhexadecylether(³H-CHE). LN formulations were mixed with mouse plasma at a final lipidconcentration of 0.75 mg/ml, followed by incubation at 37° C. At varioustime points, aliquots were taken and run over Sephadex G-50 spin columnsto remove the unentrapped prodrug (Pick, Arch. Biochem. Biophys.,212:186-194 (1981)). The derivative and lipid concentrations in theeluates were determined by UHPLC and liquid scintillation counting,respectively.

FIG. 7A shows the percent retention of TD 1 in LN, defined as thederivative/lipid (or prodrug/lipid) ratio found in the sample at aspecified time point divided by the initial drug-to-lipid ratio. BothDSPC/Chol and DPPC/Chol LN show no or little release over the 16 h timecourse of the experiment. DMPC/Chol LN released the prodrug with ahalftime of about 6 hours with 16% of the prodrug remaining entrapped 16h post injection. Comparison of the retention profiles of DSPC/Chol LNformulations loaded at 0.1 and 0.2 mg/mg shows that drug retention isnot dependent on prodrug-to-lipid ratio. In vitro release studiesperformed in mouse plasma are in good agreement with the in vivo studies(FIG. 7B). The increase in release seen with DMPC/Chol LN compared toDSPC and DPPC/Chol LN is consistent with a decrease in membranepermeability in going from DMPC, which has the shortest acyl chains(C14) to the longer chain lipids. FIG. 7C shows the in vitro retentionproperties of TD1 in DSPC/chol LN relative to other docetaxelderivatives (TD2-3 and TD7) formulated in DSPC/chol at the sameprodrug-to-lipid ratio. All derivatives were efficiently retained. TD7was released at a rate mirroring that of TD1, whereas TD2 and TD3 werereleased at a slightly faster rate (percent release is defined as 100minus percent retention). The in vitro and in vivo data demonstrate thatweak base derivatives can be efficiently retained in LN and that releaserates can be regulated by varying the lipid composition of LN carriers.

Example 13 Pharmacokinetics and In Vivo Drug Release

The pharmacokinetics (PK) of LN-encapsulated docetaxel derivatives werecompared to the PK of Taxotere™, the commercial docetaxel formulation,and derivatives formulated in the same manner as Taxotere™. Taxotere™and similarly formulated derivatives were formulated as described in theprescribing information for Taxotere™ (Sanofi-Aventis, U.S.) usingethanol/polysorbate 80/physiological saline solution to dissolve thedrug. The docetaxel derivative was encapsulated in DSPC/chol, DPPC/Choland DMPC/Chol LN (55:45 mol %) at a drug-to-lipid ratio of 0.2 wt/wtusing the ammonium sulfate loading technique. The lipid componentscontained trace amounts (0.15 μCi/mg lipid) of the lipid marker [³H]CHE,allowing monitoring of the elimination of both the prodrug and the LNcarrier from circulation.

The PK and in vivo release studies were based on 4 time points (1, 4, 8and 16 hrs) and 4 mice per time point. All formulations wereadministered i.v. via the lateral tail vein at docetaxel (or equivalentdocetaxel) doses of 20 mg/kg and volumes based on subject weight (10mL/kg). At various times, mice were anesthetized with ketamine/xylazineand blood was collected by cardiac puncture and placed into EDTAmicrotainer tubes. Animals were terminated immediately after bloodcollection. Plasma was separated from whole blood by centrifugation at1,000 g for 10 min. Plasma proteins were precipitated by the addition of150 μl of ice-cold methanol acidified with 0.1% TFA to 50 μl of plasma.The methanolic solutions were centrifuged for 30 min at 15,000×g at 4°C. to pellet the proteins and the supernatant was analyzed for docetaxeland drug derivatives by UHPLC. For LN formulations, 25-50 μl of plasmawas added to scintillation fluid (PicoFluor 40, Perkin Elmer) andanalyzed for lipid levels ([³H]-CHE radioactivity) by scintillationcounting. The percentage of prodrug remaining in LN (drug retention) wascalculated by dividing the prodrug/lipid ratios found in plasma samplesby those of the injected LN formulations, taken as 100%. Results areshown in FIG. 7B. As free docetaxel and docetaxel derivatives werecleared at much faster rates than LN-encapsulated forms, theprodrug/lipid ratios recovered from the plasma samples can be regardedas a direct indication of the amount of prodrug remaining encapsulatedin LN.

Example 14 In Vitro Anticancer Activity

The ability of the derivative to form the active drug (bioconversion)was further investigated by measuring the anticancer activity of TD 1 invitro relative to the parent compound (docetaxel). Anticancer activitywas evaluated against a panel of 3 human cancer cell lines, includingthe ovarian cancer cell line ES-2, the prostate cancer cell line PC3 andthe breast cancer cell line MDA435/LCC6 (BC Cancer Research Centre,Vancouver, BC) (Fields and Lancaster, Am. Biotechnol. Lab., 11:48-50(1993); Nakayama et al., J. Immunol. Methods, 204:205-208 (1997)).Cytotoxicity was determined using the Alamar Blue assay after a 72-hdrug exposure period. Briefly, cells were incubated in 96 well plates at37° C. for 72 hrs in the presence of varying amounts of TD1 or theparent drug (dissolved in DMSO); at the end of the incubation period,Alamar Blue solution was added to all of the wells (20 μl/well, 10% ofculture volume). The plates were returned to the incubator for 4 h;sample fluorescence was determined at λ_(ex)=530 nm and λ_(em)=590 nm.Viability was calculated according to: Cell viability(%)=(F_(plus drug)−F_(background))/(F_(minus drug)−F_(background))*100,where F_(plus drug) is the fluorescence reading in the presence of drug,F_(minus drug) the cell control in the absence of drug andF_(background) the background fluorescence (media alone). IC₅₀ values(nM) were calculated by fitting a sigmoidal curve to theconcentration-viability plot and are presented in Table 4. TD1 was asactive as docetaxel, indicating that the prodrug was readily convertedinto the active compound.

TABLE 4 In vitro cytotoxicity (IC₅₀ values) of docetaxel and docetaxelderivative. IC₅₀ (nM) IC₅₀ (nM) Cell line Docetaxel Derivative PC-3(prostate cancer) 1 0.5 MDA-MB-435/LCC6 (breast cancer) <0.1 <0.1 ES-2(ovarian cancer) 0.1 0.1

Example 15 In Vivo Anticancer Activity

The anticancer efficacy of LN-docetaxel derivative formulations wasevaluated in a subcutaneous xenograft model of human breast cancer(MDA-MB-435/LCC6) after a single bolus injection. Murine MDA-MB-435/LCC6cells were cultured in DMEM with 2 mM L-glutamine and 10% FBS at 37° C.in 5% CO₂ environment. Female RAG2-M mice were inoculated with 5×10⁶ (50μL) cells subcutaneously on the right hind flank. Once tumors reached asize of 100-150 mm³, animals were randomized into groups (6 animals pergroup) and injected with a single i.v. bolus injection of Taxotere™ at adose of 25 mg/kg or LN formulations of TD1 (DSPC/Chol, DPPC/Chol andDMPC/Chol at 55:45 mol % and a prodrug/lipid weight ratio 0.2 wt/wt) atthree different doses (31.25 mg/kg, 50 mg/kg and 110 mg/kg, which iscorresponds to 25, 40 and 88 mg/kg docetaxel). Tumor growth and animalweights were measured every third day. Tumor growth was monitored bymeasuring tumor dimensions with digital calipers and tumor volumes werecalculated according to the equation length×(width²)÷2 with the length(mm) being the longer axis of the tumor. Tumors were allowed to grow toa maximum of 700 mm³ before termination; animals with ulcerated tumorswere terminated.

The effectiveness of the treatment was assessed through comparison ofestablished parameters of anticancer activity, including: tumor growthinhibition (optimal % T/C); tumor growth delay (T−C); difference in timefor treated and control tumors to double in size; and NCI score (PlowmanJ, Dykes D J, Hollingshead M, Simpson-Herren L, Alley M C. 1997. Humantumor xenograft models in NCI drug development. In: Teicher B A, editor.Anticancer drug development guide: Preclinical screening, clinicaltrials, and approval. Totowa: Humana Press, Inc. pp 101-125). Inaddition, any drug-related deaths (within 15 days of last dose and lowtumor burden) were recorded, as well as the maximum weight loss (mean ofgroup). % T/C values and NCI scores were calculated as follows: changesin tumor volumes for each treated (T) and control (C) were calculatedfor each day tumors were measured by subtracting the median tumor volumeon the day of first treatment from the median tumor volume on thespecified observation day. The resulting values were used to calculatepercent T/C according to % T/C=(ΔT/ΔC)×100. The optimal (minimum) valuewas used to quantitate antitumor activity. An NCI score of 0 is assignedto an optimal % T/C>42 and means that the treatment is ineffective. Ascore of 1 is assigned to optimal % T/C values 1-42 and indicates tumorgrowth is inhibited (Capdeville et al., Nature Reviews Drug Discovery,1:493-502 (2002)).

The influence of lipid composition of the LN carrier on the efficacy ofencapsulated TD1 is illustrated in FIG. 8A. DSPC/Chol, DPPC/Chol andDMPC/Chol LN formulations (TD 1-to-lipid ratio 0.2 wt/wt) wereadministered at a docetaxel equivalent dose of 40 mg/kg. The DSPC/Cholformulation inhibited tumor growth most effectively, followed byDPPC/Chol and DMPC/Chol (least active) formulations. Antitumor efficacyis highly correlated with the release rate, with the formulation thatexhibits the slowest release rate being the most active.

The therapeutic activity of the DSPC/Chol formulation (TD1-to-lipidratio 0.2 wt/wt) was determined at 3 different doses (25, 40 and 88mg/kg docetaxel) in comparison to 25 mg/kg Taxotere™/docetaxel. Atequimolar doses (25 mg/kg docetaxel) Taxotere™ was slightly moreefficacious than the LN formulation. However, the LN formulation can beadministered at doses much higher than the MTD of Taxotere™. At thesedoses, DSPC/Chol LN formulations were significantly more efficacious(FIG. 8B). The most significant tumor growth suppression was observed at88 mg/kg docetaxel with optimal % T/C value of 5% and a tumor growthdelay (T−C) of 29 days compared to 9 days for Taxotere™ (Table 5). Theresults demonstrate that LN formulations are potentially much moreeffective than Taxotere™.

TABLE 5 Summary of antitumor activity and tolerability parameters of thedocetaxel derivative TD1. Anti-Tumor Activity Docetaxel T-C^(b) NCIToxicity Treatment Dose (mg/kg) % T/C^(a) (days) Score DRD^(c) MWL^(d)Taxotere ™ 25 10 9 1 0/6 −3.2 DSPC/Chol 25 55 4 0 0/6 −0.9 DSPC/Chol 4021 11 1 0/6 −2.9 DSPC/Chol 88 5 29 1 0/6 −3.8 DPPC/Chol 40 21 9 1 0/6Not observed DMPC/Chol 40 42 4 0 0/6 −2.9 ^(a)Optimal % T/C. A % T/C >42 has an NCI score of 0 (inactive) and a % T/C from 1-42 has an NCIscore of 1 (stands for tumor inhibition). ^(b)Tumor growth delay(difference in time for treated and control tumors to double in size).^(c)Drug related deaths (DRD). ^(d)Maximum mean weight loss pertreatment group in percent (%), n = 6.

Example 16 Tolerability Studies

Tolerability studies were aimed at establishing the maximum tolerateddose (MTD) and the dose range for use in efficacy studies (efficacystudies were based on a single i.v. injection). Single dose MTD studieswere performed in immune-compromised SCID/Rag2-M mice (also used for theefficacy studies) using LN-encapsulated TD1, TD1 formulated in the samemanner as docetaxel in Taxotere™, and Taxotere™. The studies were basedon administration of a single dose and relied on 3 mice/group and a doseescalation strategy based on three dose levels for the derivative andTaxotere and five dose levels for the LN formulations (DSPC/Chol 55:45mol % at a prodrug/lipid weight ratio of 0.2 mg/mg). All formulationswere injected i.v. via the lateral tail vein in a volume of 200 μl/20 gmouse.

Mice were monitored daily for signs of toxicity over a period of 14 daysfollowing drug administration. Tolerability was assessed by changes inbody weight as well as behavioral parameters. The MTD was defined as thedose that results in ˜15% loss in body weight and does not causelethality. Body weights of individual mice were measured every secondday over the course of the study. If weight loss was not a goodpredictor of tolerability, the dose where no animals needed to beterminated due to toxicity was used.

The results are summarized in Table 6. The single dose MTD of Taxotere™was 29 mg/kg while the MTD of TD1 was 16 mg/kg (MTD in docetaxelequivalents corresponding to 20 mg/kg prodrug). TD1 showed acutetoxicity (lethality) at a docetaxel equivalent dose of 20 mg/kg. Theacute toxicity appeared to be related to drug precipitation followinginjection. In contrast, LN-encapsulated TD1 (DSPC/Chol 55:45 mol % LN,prodrug/lipid weight ratio 0.2 mg/mg) was well tolerated with no signsof toxicity (no significant changes in body weight and behavioralparameters) at docetaxel equivalent doses as high as 88 mg/kg (Table 6).The MTD of the LN formulation is at least 3 times higher than that ofTaxotere™ (29 mg/kg) demonstrating that it is much better tolerated(less toxic) and thus can be administered at higher and more efficaciousdoses. Vehicle (polysorbate 80/physiological saline) alone had noadverse effects.

TABLE 6 Maximum tolerated doses (MTD) of Taxotere ™. Max Dose Wt LossClin. MTD Drug (mg/kg) (%) DRD Observations (mg/kg) Taxotere 22 −2.1 0/329 −2.7 0/3 29 36 + 1/3 LN formulation 40 −0.4 0/3 52 −3.5 0/3 64 −0.60/3 76 −1.5 0/3 88 −2.3 0/3 >88 Derivative 16 + 0/3 16 20 1/1 Acutetoxicity 24 3/3 Acute toxicity DRD: drug-related death

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed:
 1. A pharmaceutical formulation comprising a liposomalnanoparticle having an interior compartment, wherein the interiorcompartment contains a docetaxel derivative having the structure:

or a pharmaceutically acceptable salt thereof, wherein—C(═O)-(Spacer)-[N] is selected from the group consisting of:


2. The formulation of claim 1, wherein the liposomal nanoparticlecomprises a phosphatidylcholine containing saturated C₁₄-C₂₂ carbonchains.
 3. The formulation of claim 2, wherein the liposomalnanoparticle further comprises a sterol.
 4. The formulation of claim 3,wherein the liposomal nanoparticle comprises the sterol and thephosphatidylcholine in a molar ratio of from 0.1:1 to 1:1.
 5. Theformulation of claim 1, wherein the interior compartment comprises anaqueous, low pH buffer.
 6. The formulation of claim 5, wherein the lowpH buffer comprises citrate.
 7. The formulation of claim 1, wherein theinterior compartment comprises an aqueous solution containing anionophore selected from the group consisting of nigericin and A23187. 8.The formulation of claim 1, wherein the interior compartment comprisesan aqueous solution containing ammonium sulfate.
 9. The formulation ofclaim 1, wherein the ratio of the docetaxel derivative to the lipids inthe liposomal nanoparticle is from about 0.01:1 to about 10:1 by weight.10. The formulation of claim 1, wherein the lipid concentration of theformulation is from about 0.5 mg/mL to about 100 mg/mL.
 11. Theformulation of claim 10, wherein the lipid concentration is from about10 mg/mL to about 50 mg/mL.
 12. The formulation of claim 1, wherein theliposomal nanoparticle is less than about 0.05 microns in size.
 13. Theformulation of claim 1, wherein the liposomal nanoparticle is betweenabout 0.1 and 0.5 microns in size.
 14. The formulation of claim 1,wherein the liposomal nanoparticle is suspended in an aqueous externalmedium.
 15. The formulation of claim 1, further comprising one or moresubstances selected from the group consisting of a pH adjusting agent, abuffering agent, a tonicity adjusting agent, a free radical quencher,and an antioxidant.
 16. The formulation of claim 1, wherein theformulation is lyophilized.
 17. The formulation of claim 1, wherein theformulation is sterilized.
 18. The formulation of claim 1, wherein theliposomal nanoparticle has an in vivo release half-life of between about1 to about 96 hours.
 19. The formulation of claim 1, wherein: theliposomal nanoparticle is between about 0.1 and 0.5 microns in size; theliposomal nanoparticle comprises a sterol and a phosphatidylcholine in amolar ratio of from 0.1:1 to 1:1, the phosphatidylcholine containingsaturated C₁₄-C₂₂ carbon chains; the interior compartment comprises anaqueous, low-pH citrate buffer; the liposomal nanoparticle is suspendedin an aqueous external medium; the lipid concentration of theformulation is from about 10 mg/mL to about 50 mg/mL; and the ratio ofthe docetaxel derivative to the lipids in the liposomal nanoparticle isfrom about 0.01:1 to about 10:1 by weight.
 20. The formulation of claim1, wherein the docetaxel derivative has the structure:


21. The formulation of claim 19, wherein the docetaxel derivative hasthe structure: